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mblinternationalcorporation · 7 months ago

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Precision in Practice: Optimizing Cell Culture Reagents for Reliable Results

Cell culture techniques are foundational to a vast array of biological research endeavors, ranging from basic cell biology studies to drug discovery and regenerative medicine. At the heart of successful cell culture experiments lie the reagents employed, which serve as the essential components in providing the necessary nutrients, growth factors, and environmental conditions for cell growth and proliferation. In this article, we delve into the critical role of cell culture reagents and explore strategies for optimizing their use to achieve reliable and reproducible results.

Understanding Cell Culture Reagents:

Cell culture reagents encompass a wide range of substances designed to support cell growth, maintenance, and experimentation in vitro. These reagents include cell culture media, supplements, growth factors, sera, and other additives tailored to meet the specific requirements of different cell types and experimental conditions. Each component plays a crucial role in creating an environment conducive to cell viability, proliferation, and functionality.

Cell Culture Media:

Cell culture media form the basis of cell culture systems, providing essential nutrients, salts, vitamins, and buffering agents necessary for cell survival and growth. Depending on the cell type and experimental objectives, different formulations of media may be utilized, such as Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640, or Minimum Essential Medium (MEM). It is essential to select the appropriate medium composition and supplements to support the specific metabolic needs and growth characteristics of the cells under investigation.

Serum and Serum-Free Alternatives:

Traditionally, fetal bovine serum (FBS) has been a common supplement in cell culture media due to its rich source of growth factors, hormones, and proteins essential for cell growth. However, concerns regarding variability, ethical considerations, and potential contamination have led to the development of serum-free alternatives and defined media formulations. Serum-free media offer greater control over experimental conditions and reduce the risk of introducing unknown factors into cell culture systems, thereby enhancing reproducibility and reliability.

Growth Factors and Cytokines:

In certain cell culture applications, the addition of specific growth factors or cytokines is necessary to promote cell proliferation, differentiation, or function. These bioactive molecules mimic the physiological signaling cues present in vivo and play critical roles in regulating cellular processes. Researchers must carefully titrate the concentration of growth factors to optimize cell response while avoiding potential adverse effects or signaling pathway saturation.

Quality Control and Validation:

Ensuring the quality and consistency of cell culture reagents is paramount to obtaining reliable and reproducible results. Reputable suppliers employ stringent quality control measures, including batch testing, sterility testing, and endotoxin testing, to assess the purity, potency, and safety of their products. Researchers should verify the performance of newly acquired reagents through validation experiments, comparing them against established protocols and reference standards to confirm their suitability for intended applications.

Optimizing Experimental Parameters:

In addition to selecting high-quality reagents, optimizing experimental parameters is essential for achieving precise and reliable cell culture results. Factors such as temperature, pH, gas exchange, and culture vessel surface properties can significantly impact cell behavior and experimental outcomes. By systematically optimizing these parameters and standardizing experimental protocols, researchers can minimize variability and enhance the reproducibility of their results.

Temperature and Gas Exchange:

Maintaining optimal temperature and gas exchange conditions is critical for supporting cell viability and metabolic activity in culture. CO2 incubators provide a controlled environment with regulated temperature, humidity, and CO2 levels, mimicking physiological conditions conducive to cell growth. Proper calibration and routine maintenance of CO2 incubators are essential to ensure stability and consistency in cell culture experiments.

pH and Buffering:

Maintaining the pH of cell culture media within the physiological range is essential for cell viability and function. Buffering agents such as bicarbonate-based or HEPES-buffered media help stabilize pH fluctuations and maintain osmotic balance in the culture environment. Regular monitoring of pH levels and adjustment as needed are necessary to prevent pH drift and ensure optimal conditions for cell growth.

Surface Coating and Cell Adhesion:

For adherent cell cultures, the choice of culture vessel surface coating can significantly influence cell adhesion, spreading, and morphology. Substrates such as collagen, fibronectin, or gelatin facilitate cell attachment and spreading by providing ligands for cell surface receptors. Pre-coating culture dishes or plates with the appropriate substrate and optimizing coating concentration and incubation time are critical steps in promoting cell adhesion and maintaining culture integrity.

Conclusion

In conclusion, optimizing cell culture reagents and experimental parameters is essential for achieving precision and reliability in cell culture experiments. By selecting high-quality reagents, validating their performance, and optimizing experimental conditions, researchers can minimize variability, enhance reproducibility, and generate robust data. Attention to detail in reagent selection, validation, and experimental design is paramount to advancing our understanding of cellular biology, disease mechanisms, and therapeutic interventions in biomedical research. As we strive for precision in practice, the optimization of cell culture reagents remains a cornerstone of scientific excellence and discovery in the field of biology and medicine.

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vanshika393 · 1 year ago

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Fetal Bovine Serum Market Share and Demand Analysis with Size, Growth Drivers and Forecast to 2030

The latest market report published by Credence Research, Inc. “Global Fetal Bovine Serum Market: Growth, Future Prospects, and Competitive Analysis, 2016 – 2028. The global Fetal Bovine Serum Market has grown steadily in recent years and is predicted to expand at a CAGR of 5.60% between 2023 and 2030. In 2022, the market was worth USD 849.8 million, and it is predicted to grow to USD 1314.0 million by 2030.

Introduction: Unveiling the Significance of Fetal Bovine Serum

Fetal bovine serum is a vital component in cell culture, primarily due to its rich composition of growth factors, hormones, and other essential nutrients. It serves as a nutritional source for cultured cells, enabling their growth, proliferation, and maintenance. Researchers rely on FBS to cultivate a wide range of cell types, making it indispensable in various fields, including pharmaceuticals, biotechnology, and academic research.

Market Dynamics: Exploring Growth Factors

1. Increased Demand for Biopharmaceuticals

The pharmaceutical industry has witnessed a paradigm shift towards biopharmaceuticals. This shift can be attributed to the rising prevalence of chronic diseases and the need for innovative therapies. As a result, the demand for FBS has surged, as it plays a pivotal role in the production of biopharmaceuticals, including vaccines, monoclonal antibodies, and cell-based therapies.

2. Advancements in Cell-based Research

Cell-based research is at the forefront of scientific innovation. FBS serves as the cornerstone of cell culture, enabling scientists to conduct experiments and develop new treatments for various diseases. Its applications extend to cancer research, regenerative medicine, and drug discovery, driving the market's growth.

Fetal Bovine Serum Market Major Challenges and Risks

Supply Chain Vulnerabilities: The primary source of FBS is the blood of unborn calves, which makes it susceptible to supply chain disruptions due to factors like disease outbreaks in cattle, regulatory changes, or natural disasters. These disruptions can lead to shortages and price volatility.

Ethical Concerns: There are ethical concerns related to the use of FBS, as it involves the collection of blood from unborn calves, which raises animal welfare and cruelty issues. These concerns can result in regulatory restrictions or push for alternative serum-free culture media.

Regulatory Changes: Regulations surrounding the collection and use of FBS can vary between countries and regions. Changes in regulatory requirements can impact the availability and cost of FBS, as well as necessitate costly adjustments for producers and users to comply with new standards.

Contamination and Quality Control: Ensuring the quality and safety of FBS is crucial in cell culture applications. Contamination with pathogens, mycoplasma, or other undesirable elements can pose a significant risk to experiments and processes. Quality control and testing are essential but can be costly.

Browse 247 pages report Fetal Bovine Serum Market By Grade (Cell Culture Biopharmaceuticals Production Vaccine Production In Vitro Fertilization (IVF Diagnostics and Research) By Distribution Channel (Research Institutes and Laboratories Biotechnology Companies Pharmaceutical Companies Academic Institutions) By Grade (Premium Superior Standard)- Growth, Future Prospects & Competitive Analysis, 2016 – 2030- https://www.credenceresearch.com/report/fetal-bovine-serum-market

Market Trends

1. Advent of Serum Alternatives

While Fetal Bovine Serum has been a cornerstone of cell culture, there is a growing trend towards the development of serum alternatives. These alternatives aim to address ethical concerns and reduce the risk of contamination, which can be advantageous in specific research scenarios.

2. Asia-Pacific Emerging as a FBS Hub

The Asia-Pacific region is witnessing remarkable growth in the FBS market. Factors such as the increasing number of research institutions, availability of skilled labor, and cost-effective production have made the region a key player in the global FBS industry.

Some of the major players in the market and their market share are as follows: Danaher (Cytiva), Merck KGaA (Sigma Aldrich), HiMedia Laboratories Pvt. Ltd, Bio-Techne, Sera Scandia (Biowest), Sartorius (Biological Industries), Atlas Biologicals, Rocky Mountain Biologicals, PAN-Biotech.

Key Segments

By Grade

Cell Culture

Biopharmaceuticals Production

Vaccine Production

In Vitro Fertilization (IVF)

Diagnostics and Research

By Distribution Channel

Research Institutes and Laboratories

Biotechnology Companies

Pharmaceutical Companies

Academic Institutions

Why to Buy This Report-

The report provides a qualitative as well as quantitative analysis of the global Fetal Bovine Serum Market by segments, current trends, drivers, restraints, opportunities, challenges, and market dynamics with the historical period from 2016-2020, the base year- 2021, and the projection period 2022-2028.

The report includes information on the competitive landscape, such as how the market's top competitors operate at the global, regional, and country levels.

Major nations in each region with their import/export statistics

The global Fetal Bovine Serum Market report also includes the analysis of the market at a global, regional, and country-level along with key market trends, major players analysis, market growth strategies, and key application areas.

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About Us -

Credence Research is a viable intelligence and market research platform that provides quantitative B2B research to more than 10,000 clients worldwide and is built on the Give principle. The company is a market research and consulting firm serving governments, non-legislative associations, non-profit organizations, and various organizations worldwide. We help our clients improve their execution in a lasting way and understand their most imperative objectives. For nearly a century, we’ve built a company well-prepared for this task.

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karanresearchlayer · 1 year ago

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CHO Cell Culture Media Market 2030 Has Huge Growth In Industry

CHO (Chinese Hamster Ovary) cell culture media are specifically formulated nutrient solutions used to support the growth, maintenance, and production of CHO cells in laboratory and industrial settings. CHO cells are commonly used in biopharmaceutical production for the expression of therapeutic proteins. Here is some information about CHO cell culture media:

For Download Sample Report Click Here: https://www.marketinforeports.com/Market-Reports/Request-Sample/520660

Basal Media: Basal media form the foundation of CHO cell culture media and provide essential nutrients and growth factors necessary for cell growth. They typically contain a balanced mixture of amino acids, vitamins, inorganic salts, glucose or other energy sources, and buffering agents to maintain pH stability. Basal media are usually supplemented with serum or serum substitutes, such as fetal bovine serum (FBS) or chemically defined serum-free supplements, to provide additional nutrients and growth-promoting factors.

Serum and Serum-Free Media: Traditionally, CHO cell culture media have utilized serum, such as FBS, to enhance cell growth and productivity. However, serum can introduce batch-to-batch variability and potential contamination risks. To overcome these challenges, serum-free media formulations have been developed. These media contain defined components that can support CHO cell growth and protein production without the use of animal-derived serum. Serum-free media often incorporate recombinant growth factors, hormones, and specific nutrients to mimic the growth-promoting properties of serum.

Feed Media: CHO cell cultures often require supplementation with feed media during the production phase to enhance cell viability, productivity, and protein quality. Feed media provide additional nutrients, such as amino acids, vitamins, and energy sources, to sustain the cells during the production of recombinant proteins. Feed media are typically added periodically or continuously to the culture to maintain optimal conditions for protein synthesis and prevent nutrient depletion.

Specialized Media: Depending on the specific requirements of CHO cell lines or the desired outcomes of the production process, specialized media formulations may be used. These specialized media can include chemically defined media, animal-component-free media, or media optimized for specific CHO cell lines or expression systems. These formulations are designed to provide precise control over the culture conditions and maximize protein yield and quality.

Buffer Systems: CHO cell culture media often incorporate buffering systems to maintain a stable pH within the optimal range for cell growth. Common buffer systems used include bicarbonate, phosphate, or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The choice of buffer system depends on the specific experimental setup and requirements.

It’s important to note that the composition and formulations of CHO cell culture media can vary among different manufacturers and laboratories. Optimization of media components and culture conditions is critical for achieving high cell viability, productivity, and protein quality in CHO cell cultures. Specific media formulations are typically tailored to the specific needs of the cell line and the intended application, and they are often developed through a combination of empirical optimization and scientific expertise.

#CHO Cell Culture Media Market

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rnomics · 5 years ago

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Viruses, Vol. 12, Pages 139: Optimized Hepatitis E Virus (HEV) Culture and its Application to Measurements of HEV Infectivity

Hepatitis E virus (HEV) is a major concern in public health worldwide. Infections with HEV genotypes 3, 4, or 7 can lead to chronic hepatitis while genotype 1 infections can trigger severe hepatitis in pregnant women. Infections with all genotypes can worsen chronic liver diseases. As virions are lipid-associated in blood and naked in feces, efficient methods of propagating HEV clinical strains in vitro and evaluating the infectivity of both HEV forms are needed. We evaluated the spread of clinical strains of HEV genotypes 1 (HEV1) and 3 (HEV3) by quantifying viral #RNA in culture supernatants and cell lysates. Infectivity was determined by endpoint dilution and calculation of the tissue culture infectious dose 50 (TCID50). An enhanced HEV production could be obtained varying the composition of the medium, including fetal bovine serum (FBS) and dimethylsulfoxide (DMSO) content. This increased TCID50 from 10 to 100-fold and allowed us to quantify HEV1 infectivity. These optimized methods for propagating and measuring HEV infectivity could be applied to health safety processes and will be useful for testing new antiviral drugs. http://bit.ly/2tBeLSV

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we-future-first · 5 years ago

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[in-depth] How it's made: the science behind cultivated meat, part 4D: cell culture medium for seafood

The /r/Futurology subreddit frequently features highly upvoted posts on cultivated meat, reflecting the media attention and public interest that has followed the industry. There are many introductory resources to how cultivated meat is produced and what its benefits may be, however, there are no comprehensive resources that fully inform those interested in learning more. Below you’ll find the seventh post in a multi-part series that walks through the science driving the innovative technology of cultivated meat. These posts are intended to be educational but lengthy and best understood by those with science backgrounds.

Please check out the previous posts linked below. Writing can also be viewed altogether here (recommended).

Series I: Cell Lines

Series II: Bioprocessing

Series III: Bioengineering A and B

Series IV: Cell culture media A and B

Series V: Differentiation and Final products

Series VI: Impact (environment, human health, food security, animal welfare)

Introduction

Although aquatic animal cell lines from embryonic and other lineages have been successfully established and studied, this field is in its infancy compared to cell culture from terrestrial vertebrates. In particular, a full-scale optimization of media composition has not been performed for any aquatic species. Here, previous cell culture studies from aquatic species are discussed in order to glean insights regarding what components are likely or unlikely to be necessary in an optimized, animal component-free formulation suitable for producing cultivated seafood. Comparisons of in vivo nutritional requirements and meat composition for aquatic and terrestrial species are also referenced, although due to potential limited translatability between in vivo and in vitro characteristics, such comparisons should be considered qualitative in nature.

Media composition is considered separately for fish embryonic stem (ES)-like cells, other fish cells, and cells from crustaceans. The term ES-like cells is used because while some of the lines mentioned have been shown to express multiple ESC markers and to exhibit other stem cell-like characteristics such as forming embryoid bodies, others have been less fully characterized. ES-like cells should be understood to include both ESCs and other cells that may be partially differentiated.

Optimizing culture media for aquatic species is a tractable problem, made easier by the plethora of research already available from terrestrial species. However, much work is still needed to actually perform systematic optimizations of the concentrations of various components for teleost fish, crustaceans, and other aquatic taxa. Still further optimization will be required to fine-tune media composition for specific species and cell types. Specific strategies for optimization will be explored in the next part of this series.

Comparison of culture media for ES-like cells from fish and mammals

Compared to mammalian cell culture, much less is known about the in vitro requirements of fish cells. The most common media formulation used to grow fish ES-like cell lines was originally developed for medaka cells, a small fish commonly used as a research model organism.1 This formulation, called ESM1 in the original publication, includes not only fetal bovine serum (FBS) but also fish serum and fish embryo extract. Two recombinant human growth factors, FGF2 (also known as bFGF) and LIF, are included as well. A comparison of ESM1 and some of its derivatives with the serum-free Essential 8 (commonly known as E8)2 and B83 formulas optimized for human ESC and iPSC culture reveals several similarities and differences (Table 1).

All formulations are based on common basal media such as DMEM, L-15, or a mixture of one or both of these with F12, and contain buffers, some form of antioxidant or reactive oxygen species scavenger, and a source of selenium ions. They contain either the same or similar recombinant growth factors. LIF, included in ESM1, and TGFβ/NODAL, included in E8, might play a similar role to one another, as it has been shown that treatment with TGFβ induces robust expression of LIF in glioblastoma cells.4 Similarly, treatment of explants from the developing rat kidney with either LIF or TGFβ induced expression of LIF as well as a modest increase in expression of TGFβ.5 NRG1, included only in B8, enhances growth but is not required.3 E8 and B8 contain two components with no direct analog in ESM1: transferrin and insulin. In addition, E8 and B8 contain substantially higher concentrations of sodium selenite and FGF2 than ESM1. ESM1, unlike E8 and B8, contains FBS, fish serum, and fish embryo extract. These animal-derived components serve as sources of transferrin, insulin, selenium, and FGF2 (or analogs thereof), and thus at least some of these components will likely be required in order to produce optimized serum-free media.

Table 1: Comparison of E8, B8, and several media formulations used to culture fish ES-like cells. Components not separated by a horizontal line are those likely to substitute for one another, due to playing a similar function or interacting with similar receptors or signaling pathways. Bold borders indicate that the publication reported directly testing the effect of this component on fish cell growth and finding a positive effect. Double borders indicate components (besides basal media) shown to be essential, rather than simply growth-enhancing, although the essentiality of TGFβ is apparent only in long-term growth assays.3 Color scales for β-mercaptoethanol, selenium, FGF2, FBS, and fish serum facilitate visualization of concentration differences between formulations. Question marks indicate that the publication did not state a concentration for the reported component. Note that the B8 media formulation uses the thermostable variant FGF2-G3, so its effective concentration in the media may be higher than it appears relative to other publications. Antibiotics and buffers are not shown. Also see Dash et al. 2010.6

What is not needed for culture of fish ES-like cells?

Most publications using fish ES-like cells employ media formulations based on ESM1. The only successful instance of serum-free culture of fish ES-like cells was in medaka, and was achieved by supplementing DMEM with IGF2.7 This formulation, while able to support growth, did not perform as well as the control formulation, which contained all the same components as ESM1 except for LIF, and was not tested for its ability to support long-term culture. An essential step in identifying optimized media formulations for use in fish stem cell culture will therefore be the identification of the additional components of serum and embryo extract that are necessary and sufficient for optimal growth of fish stem cells in animal-free media. The composition of naturally-occurring serum is described in detail in part B of this series. Identification of a set of components that can support robust growth of fish ES-like cells in the absence of serum will require careful testing of a number of candidates and optimization of their respective concentrations.

Some components of ESM1 are frequently omitted in other studies, and therefore can probably be deprioritized in attempts to find optimized animal-free formulations. The growth factor LIF was used only during early passages in the original publication,1 and is one of the components most commonly omitted in other studies,8,9,10,11,12,13,14 suggesting that it may not be essential at least in some species or lines. In an ES-like line from Indian major carp, LIF was shown to stimulate growth and to help maintain an undifferentiated state, although both effects were quite modest when tested in the presence of other growth factors.6 ESM1 contains glutamine, pyruvate, and non-essential amino acids (NEAA) as additional nutrient sources, but cells from turbot, olive flounder, barramundi, and haddock have been successfully cultured without these supplements.11,12,13,14 Glutamine was also omitted in studies of ES-like cells from Japanese sea bass and red seabream,15,1600570-7) consistent with the glutamine independence of mammalian ESCs mentioned in part A.17

Two studies are notable outliers: ES-like cell lines from barramundi13 and haddock14 were both successfully cultured in L-15 with 15-30% FBS. However, systematic comparisons to media containing recombinant growth factors, fish serum, and embryo extract were not performed, so it cannot be concluded that L-15 with FBS was sufficient for optimal proliferation. Although elimination of FBS needs to be the eventual goal when it comes to cultivated meat and seafood, the fact that some fish ES-like cells from common food species will grow in the absence of fish serum or embryo extract is encouraging because it suggests that ES-like fish cells recognize and require the same growth factors that have already been well characterized for serum-free culture of non-aquatic cell lines. Unfortunately, the three ESM1 components that will be essential to replace for cultivated seafood applications are consistently used across all (FBS) or most (fish serum, fish embryo extract) studies of ES-like cells from food-relevant fish species.

Requirements for serum and growth factors

It is unclear to what extent the necessity of each of these components has been systematically tested in most of the above-mentioned studies. The importance of FGF2 and FBS in cod18 and of FGF2, FBS, and fish serum in turbot11 were experimentally demonstrated by subtracting these individual components from the complete medium. The reverse strategy — adding specific components to a minimal medium — revealed that, for carp ES-like cells, fish embryo extract and FGF2 were the most capable of supporting growth when added individually to basal media containing NEAA and sodium pyruvate, while FBS, LIF, and fish serum had weaker effects.6 Differences in growth factor concentration requirements both between fish and terrestrial cells and between fish cells of different species may result not only from the intrinsic growth factor requirements of these cells but also from differences in growth factor stability at different temperatures.19 Since serum is a complex mixture of proteins, peptides, and other molecules, identifying the minimal set of components that can substitute for serum will be a key challenge. Below, several candidate serum components that could be necessary for optimal survival and proliferation of fish ES-like cells are discussed. Many of these are routinely used for serum-free culture of mammalian ESCs, and were discussed in further detail in part B of this series.

Role of insulin and insulin-like growth factors in fish

Although zebrafish have been demonstrated to respond to human insulin and to develop insulin resistance in a similar manner to mammals when exposed to high doses,20 insulin’s effects on fish stem cell proliferation are thus far unclear. However, the related insulin-like growth factors IGF1 and IGF2 have been better studied in this context. Moderate levels of IGF1 supplementation led to a modest increase in growth of a fibroblast-like catfish cell line21. The medaka ESC line HX1 showed a dramatic change in morphology and loss of expression of stem cell markers when transitioned from media similar in composition to ESM1, described above, to DMEM alone. Both of these effects were largely rescued when the cells were transitioned to DMEM containing IGF2.7 Further, in the same study, DMEM with IGF2 was able to support derivation of blastomeres, although slightly less efficiently than control media. In contrast, IGF1 showed only a very weak ability to stimulate proliferation and was unable to support maintenance of an undifferentiated state in Indian major carp.6 It is unclear whether this reflects a difference between IGF1 and IGF2 or a species difference. Insulin, because of its demonstrated ability to support growth of mammalian ESCs, and/or IGFs, because of their ability to improve survival and proliferation in fish, are likely to be necessary components of any serum replacement strategy. It is unclear to what extent insulin, IGF1, and IGF2 may be able to substitute for one another, since all three are known to interact with multiple receptors and to overlap in their receptor specificities, albeit with substantially different affinities.22,23,24

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Figure 1. Supplementation of basal media with increasing amounts of IGF2 (c-f) results in a partial rescue of the morphological changes of medaka ESCs transitioned from serum-containing media (a) to unsupplemented serum-free media (b). IGF2 also partially rescues the viability of these cells (g, measured as mean fluorescence intensity, MFI, of AlamarBlue). From Yuan & Hong 2017.7

Role of transferrin and iron in fish

Transferrin is involved in the regulation of iron metabolism in both fish and other vertebrates by binding to free iron ions. This both prevents them from causing formation of reactive oxygen species, and simultaneously increases the availability of iron at appropriate sites within cells25. As in other vertebrates, iron is an essential micronutrient in fish. The dietary iron requirement for Atlantic salmon was estimated as 60 mg iron per kg of diet, similar to the 50 mg/kg of diet recommended for beef cows, with iron-deficient salmon developing hypochromic microcytic anemia. It is important to bear in mind that these numbers are based on the whole organism, and reflect the needs of many cell types. Raw Atlantic cod contains 0.38 mg of iron per 100 g serving, lower than raw steak, 1.85 mg per serving, or raw chicken, with 0.92 mg per serving in light meat and 1.22 mg per serving in dark meat. Other fish are more comparable to terrestrial species, such as raw skipjack tuna, 1.25 mg per serving. Such quantitative differences in iron requirements or accumulation may reflect species differences in cellular requirements for iron, differences in how easily iron is absorbed, or some combination of these two factors. However, the fact that both iron and its carrier transferrin appear to behave similarly between fish and terrestrial vertebrates suggests that they may be similarly required in cell culture media, albeit possibly at different concentrations. In other words, addition of transferrin to fish stem cell media is likely to be beneficial. DMEM already contains ferric nitrite, but the optimization of the iron concentration specifically for culture of fish ES-like cells could also yield improved results.

Selenium requirements in fish

Selenium is present in E8 media at a concentration of 80 nM2. This concentration may not be high enough for maximal growth; further optimization of E8 led to the B8 formulation, in which selenium is included at a concentration of 116 nM3. In contrast, concentrations between 2 nM and 8 nM have been used for culture of fish ES-like cells, and some studies use no defined selenium source at all (see Table 1). It is possible that cellular requirements for selenium are different between fish and mammals, so it is worth considering what is known about the requirements for selenium in fish. Selenium requirements vary substantially across fish species, from 0.03 mg/kg of diet recommended for salmonids to 0.7 mg/kg of diet for juvenile grouper. The recommended minimum selenium dosage for beef cows is 0.1 mg/kg of diet, within the range recommended for various fish species. Fish also accumulate selenium within their muscles. The selenium content for raw Atlantic cod is higher, at 33.1 μg per 100 g serving, than that of raw steak, 21.1 μg per serving, or raw chicken, whether light meat, 17.8 μg per serving, or dark, 13.5 μg per serving. As with transferrin, there is no obvious reason why selenium would not be required for fish stem cell media in similar concentrations as for cells from terrestrial vertebrates. Identifying the optimum level for any given fish species should be part of any serum replacement strategy.

Choice of buffering agent

Standard practice in culturing fish ES-like cell lines is to maintain cultures under ambient CO2 conditions and to rely on HEPES as the primary buffering agent. One study found that chick ESC cultures were negatively impacted by the presence of HEPES or other zwitterionic buffers,26 but it is unknown whether the same effects are likely in fish cells, and if so at what concentrations. Systematic testing of various buffering agents might be helpful to find ideal culture conditions for fish ES-like cells.

Implications for serum-free culture of fish ES-like cells

The combination of factors that will allow for replacement of serum and other animal-derived components for fish ES-like cells while supporting optimal proliferation rates, and their optimal concentrations, is currently unknown. A good starting point will be to test the components shown to be capable of replacing serum in terrestrial vertebrate cell culture: selenium, transferrin (potentially in conjunction with adjustment of iron concentrations), insulin, and various growth factors. IGF2 in particular is a strong candidate, as its addition to the media can support serum-free culture of medaka ESCs, albeit with a reduction in growth rate compared to conventional media.7 It is likely that the addition of other components together with IGF2 will allow for high and sustained proliferation rates of fish ES-like cell cultures without animal-derived ingredients.

Media for non-embryonic fish cells

In general, published media formulations for non-embryonic cells from fish are somewhat simpler than most of those discussed above, although they also typically contain FBS and suffer from a similar lack of systematic optimization. Primary cultures from muscle (presumably containing a mixture of myocytes, myosatellite cells, and other cell types) of rainbow trout and gilthead sea bream have been initiated using DMEM with 15% horse serum, and subsequently cultured in 10% FBS.27,28,29 Under these conditions, cells fused and eventually formed large myotubes over the course of 10 to 15 days.28,29 A continuous cell line with an epithelial-like morphology isolated from southern bluefin tuna was successfully maintained over 8 months in L-15 containing 10-20% FBS.30 Concentrations of FBS below 10% resulted in reduced proliferation rates. Human FGF2 was included during isolation and early passages, but whether it affected survival or proliferation was not assessed. Serum-free culture of ovarian cells from channel catfish has been achieved by gradual reduction in the serum concentration present in the media.31 Although this cell type is not likely to be relevant to cultivated meat, the strategy of gradually adapting cells to serum-free conditions may be applicable to other cell types.

Of note, zebrafish and some other fish species lack the gene for inositol-3-phosphate synthase. As a result, inositol has been considered as an essential nutrient in models of zebrafish metabolism,32 whereas it is non-essential in mammals. Myo-inositol has been identified as a dietary requirement for optimal growth in carp, tilapia, and parrot fish,33,34,35 although it is worth noting that only the latter species lacks the inositol-3-phosphate synthase gene. In the absence of exact measurements of the activity of these various homologues, it is worth keeping in mind that optimization of inositol levels may be an important step in developing optimized basal media for fish, and that this optimization may need to be performed on a species-by-species basis.

Amino acid requirements in fish

Amino acid requirements in terrestrial species were discussed in part A of this series. In vivo, the same amino acids are essential in most tested fish species36 as in humans. The exception is arginine, which is essential in vivo in fish but only essential in vitro in human cells. Quantitative dietary amino acid requirements of a number of fish species may serve as useful guides for researchers attempting to optimize cell culture media for particular fish species, but it is unclear how direct the translation between quantitative in vivo and in vitro requirements will be. Amino acid requirements of various Mediterranean fish species37 and of salmonids38 have been reviewed in detail. Which amino acids, if any, are required in vitro but not in vivo in fish species is unknown. The successful growth of medaka ESCs in DMEM supplemented with only IGF27 suggests that the in vitro essential amino acids are likely similar between fish and mammals, although this leaves room for differences in optimal concentrations. Amino acid concentration may be an important factor in species-specific optimization of media for both embryonic and non-embryonic fish cell lines, especially in cases where minimizing the cost of media is essential. However, it may not be necessary in order to achieve the goal of finding a media formulation that works reasonably well for culturing cells from most fish species.

Osmolality

Teleost fish maintain the osmolality of their extracellular fluids within a fairly narrow range of around 300 mOsm/kg by actively either taking in (for freshwater fish) or excreting (for marine fish) salt.39 This is close to DMEM’s osmolality of between 310 and 360 mOsm/kg. Consistent with this, the fish cell culture studies cited above do not deliberately adjust osmolality, and media used to culture cells from Nile tilapia,9 which is restricted to fresh or brackish water, is of a similar formulation to those used for cells from marine species. The exception to this is fish which are osmoconformers, meaning that the osmolality of their extracellular fluids matches that of their environment. Osmoconformers are rare among aquatic vertebrates, but include elasmobranchs and hagfish.39 Mesenchymal stem cells from shark embryos were successfully cultured in media with a lower solute concentration (in this case expressed as osmolarity) than adult cartilaginous fish plasma, an observation that may be related to the fact that the fluid within the egg contains a lower solute concentration than the surrounding seawater.40 Culture of cells from osmoconforming species may thus require an explicit consideration of osmolality as it relates to both species and the life stage from which the cell line was derived.

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Figure 2. Both freshwater (a) and saltwater (b) fish actively regulate ion transport in order to achieve the correct osmolality in their tissues. Wikipedia.

Media for crustacean cell culture

In general, the in vivo requirements for most minerals between fish and aquatic crustaceans are fairly similar, with some important differences.41 Additionally, it may be possible to learn some lessons from insect cell culture, due to the close evolutionary relationship between these groups. As in vertebrates, selenium is thought to be required by shrimp and potentially other crustaceans, although a precise value for the requirement has not been reported. Although evidence for regulated transport and storage of iron has been observed in crustaceans, no evidence for iron deficiency in shrimp has been reported. This is consistent with the fact that crustaceans primarily rely on the copper-containing hemocyanins rather than iron-containing hemoglobin for oxygen transport. Indeed, most malacostracans (a group that contains both shrimp and crabs) lack hemoglobin entirely.42 Consistent with this, the dietary copper requirements for shrimp may be between 6-fold and 35-fold higher than those for fish, depending on the species.41 It is unclear whether crustaceans’ lower iron and higher copper requirements reflect only differences in oxygen transport mechanisms or if requirements of individual cells will also follow the same trend under culture conditions. Besides iron, the only minerals recommended for inclusion in feed formulations for fish but not for shrimp or lobster are iodine and manganese.41 However, these are most likely required only for the proper functioning of particular organs or systems, rather than generally required by cells (whether fish or land animals), and are not included in the standard DMEM formulation. In summary, iron and copper deserve special attention when adapting media formulations from fish to crustaceans because of their vastly different roles in these organisms. However, oxygen transport, the role for which these minerals are best appreciated, works very differently in culture compared to in vivo, so it is unclear how well whole-organism dietary requirements will translate to requirements of single cells.

Many attempts have been made at culturing cells from shrimp, with some success, yet continuous cell lines have not yet been reported in the academic literature. However, the wealth of studies performed on primary cells provides some insights into what media formulations show promise.43 In general, it seems that shrimp cells are adaptable to a variety of basal media formulations, but which is the most successful seems to be dependent on the cell type in question. Osmolalities used were higher than that of DMEM, ranging from 472 to 760 mOsm/kg. FBS has generally been found to support growth of shrimp cells, but may not be sufficient for optimal growth. Various extracts from shrimp or related species are often, but not always, helpful. FBS and chitosan both supported growth of cultures initiated from shrimp embryos, and a combination of FGF2 and IGF2 further stimulated proliferation.44 An extensive review of cell culture studies in shrimp has been published elsewhere,43 and includes discussion of both basal media formulations as well as supplements such as FBS and various shrimp extracts. Despite the challenges reported in establishing shrimp cell lines, the shrimp dumplings demonstrated by Shiok Meats in early 2019 point to the feasibility of culturing shrimp cells for the purpose of creating a cultivated food product.

Primary cells from crab hepatopancreas were successfully cultured in 3x concentrated L-15 buffer reconstituted in artificial seawater.45 Viability of these cells was reduced in the presence of FBS or various crab extracts. However, these negative effects of FBS are likely cell type-dependent rather than species-dependent. Primary crab hemocytes) grew well in 2x concentrated L-15 with amino acid-sugar supplement and 15% FBS.46 Similarly, 2x L-15 with 10% FBS was successfully used to grow crab eyestalk neurons, whereas 5% FBS was found to be insufficient to support survival.47

Conclusion

Optimized, low-cost, animal component-free media formulations for use with seafood-relevant species are desperately needed in order to move cultivated seafood from a promising idea to an economically viable solution to the environmental, ethical, and food security problems associated with current methods of seafood production. Fortunately, there is a wealth of data on the nutritional needs of seafood species and their biological differences from common terrestrial species raised for meat. In addition, successful optimization projects for mammalian cells3 can provide a roadmap for those attempting similar goals in seafood species. Serum-free culture of medaka ESCs has been demonstrated,7 although further optimization is needed to reduce costs, increase proliferation rates, and find formulations adapted to seafood-relevant species. These studies will take time and effort, but there is a clear path ahead towards the optimized media formulations needed to make cultivated seafood a reality. The potential effects of media composition on nutritional and organoleptic properties of cultivated seafood products, independently of differences in cellular proliferation, will be discussed in Series V for both seafood and terrestrial meat.

About / Disclosure

Claire Bomkamp, Ph.D. (/u/claire_bomkamp) is the author and is employed by The Good Food Institute, a 501(c)3 nonprofit that serves as a think-tank and accelerator for the plant-based and cultivated meat fields.

Special thanks to Inayat Batish for providing helpful suggestions and input!

Feel free to ask anything about the science discussed or how to get more involved in the future of food. Many questions will additionally be addressed in upcoming discussion topic series by my colleague Elliot Swartz, Ph.D. (/u/e_swartz)! General comments and suggestions for additions are welcomed.

submitted by /u/goodfoodinstitute [link] [comments] source https://www.reddit.com/r/Futurology/comments/g9m1m6/indepth_how_its_made_the_science_behind/

#Future #Futuristic #Future oriented

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healthtimetaylor · 5 years ago

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Curcumin supplementation has favorable effect on metabolic markers and anthropometric parameters in patients with NAFLD.

PMID: Complement Ther Med. 2020 Jan ;48:102283. Epub 2019 Dec 17. PMID: 31987259 Abstract Title: The effects of curcumin supplementation on liver function, metabolic profile and body composition in patients with non-alcoholic fatty liver disease: A systematic review and meta-analysis of randomized controlled trials. Abstract: BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Curcumin is the anti-inflammatory, antioxidant, anti-diabetic and also anti-hyperlipidemia agent and uses as herbal medicine for treating liver diseases.OBJECTIVE: The present systematic review and meta-analysis was conducted to investigate the effects of curcumin supplementation on metabolic markers and anthropometric parameters in patients with (NAFLD).METHODS: PubMed, Embase, Scopus, Web of Science and Cochrane Library were systematically searched to identify relevant randomized controlled trials (RCTs) investigating the effects of curcumin supplementation on the arms of this study in patients with NAFLD up to September 2019. Mean difference (MD) was pooled using a random effects model. Potential publication bias was assessed using Egger's weighted regression tests.RESULTS: After excluding irrelevant records, 9 RCTs included in this meta-analysis. Pooled results of included studies indicated a significant reduction in alanine transaminase (ALT), aspartate transaminase (AST), serum total cholesterol (TC), low density lipoprotein (LDL), fasting blood sugar (FBS), HOMA-IR, serum insulin and waist circumference (WC), but not in serum triglyceride (TG), high density lipoprotein (HDL), HbA1c, body weight and body mass index (BMI) following curcumin supplementation. Additionally, age- and baseline TC-based subgroup analysis indicated a significant reduction in TG and also duration- and dosage-based showed a significant change in BMI.CONCLUSION: The current study revealed that curcumin supplementation has favorable effect on metabolic markers and anthropometric parameters in patients with NAFLD.

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journalmaterialscience-blog · 5 years ago

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Passive Diffusion Processes Govern the Initial Stages of Cell Penetration into Electrospun Scaffolds Abstract We explore how the passive the diffusion of cells and active migration events interact with scaffold structure to modulate the extent to which electro spun PCL scaffolds are infiltrated by dermal fibroblasts in tissue culture. The data indicate that passive processes predominate during the first 24hr of culture. Infiltration was observed to a nearly equal extent in scaffolds produced by air impedance electro spinning in the presence and absence of air flow through the mandrel pores, demonstrating that electrostatic edge effects play a substantial role in producing the regional changes in fiber density that typify this fabrication technique. We conclude that cells preferentially sieve across the Z axis through these macro pores; however, a similar mechanism appears to be at work in scaffolds produced by conventional electro spinning techniques. The passive sieving hypothesis is supported by two lines of experimental evidence. First, a sub-population of cells rapidly populate the deeper recesses of the electro spun scaffolds over the first 24hr of culture, subsequent penetration is nearly non-existent. Second, even cells killed by Para formaldehyde incubation are observed to penetrate up to 300um deep into these scaffolds. Cyclic exposure to trypsin at 24hr intervals dramatically increased the number of cells that infiltrated electro spun scaffolds. Go to Introduction The inability to rapidly seed and populate electro spun tissue engineering scaffolds at high cell density in tissue culture represents a long recognized limitation to this emergent technology. This, despite the interconnected nature of pores that are present in these scaffolds. A broad spectrum of electro spun scaffolds composed of natural [1] synthetic polymers [2,3] and blends of natural and synthetic polymers [4] are extensively infiltrated by inflammatory, interstitial and vasculogenic cells when they are implanted in situ [5]. Cell infiltration is likely far more efficient in this setting due to a variety of variables including the presence of an intact immune response and the penetration of soluble of grow factors from the surrounding environment. The environmental factors that lead to the penetration of cells into implanted electro spun scaffolds have not been fully defined or even remotely reproduced in tissue culture. A fundamental objective of the classical tissue engineering paradigm is to produce tissues in vitro that can ultimately be used to replace or augment the function of damaged, diseased or otherwise missing organs. To fulfill this promise, regardless of the identity of the scaffold, strategies to fully populate tissue engineering scaffolds in vitro must be developed. Experiments in tissue culture environments that are nominally 3D in nature demonstrate that scaffold and fiber stiffness, fiber density, cell-matrix adhesion properties and proteolytic events all serve to modulate the rate at which cells can migrate [5-8]. In typical experiments, velocity is measured in cells moving in the X-Y orientation on tissue culture plastic coated with fibers of varying density and or physical properties. While revealing, this approach fails to capture the behavior of cells in the unique 3D environment of an electro spun matrix and it might be more accurate to state these experiments define the variables that govern migration rates on a 2D surface exhibiting complex topographical features (i.e. the fiber bundles) rather than a 3D environment. On such a surface there is virtually no depth in the Z direction where pore size and geometry will represent a major impediment to motility [9]. In a truly 3D environment cells are surrounded on all sides by the constituent elements of the surrounding matrix, regardless of its composition. This consideration is critically important in a 3D electro spun scaffold that lacks guidance cues to promote the migration in the Z direction [10] the fibers of these scaffolds are largely arrayed into layers that lie parallel with the dorsal surface of the construct. Under base line conditions the prototypical tissue culture environment appears to lack the appropriate cues necessary to drive cells to penetrate into the deeper regions of a 3D electro spun matrix. Cells seeded onto the external surfaces of these structures clearly migrate and proliferate much like they do on a fiber-coated, nominally 3D surface. However, penetration is largely limited to a few cell diameters in depth on almost all types of electro spun surfaces composed of physiologically sized fibers. Once the cells have entered the scaffold they appear to reach a thermodynamic equilibrium and without further impetus fail to penetrate into the deeper regions of the scaffold. In the present study we explore the processes that govern cell penetration into conventional electro spun scaffolds and those produced by air impedance electro spinning [11]. Air impedance electro spinning uses a hollow mandrel engineered with a series of pores as a collecting target. Air is introduced at various velocities into the center of the mandrel and then flows out of the pores. The air flow is designed to inhibit the deposition of fibers in the vicinity of the pores and thereby reduce the local density of the scaffold in a regional fashion in order to facilitate cell penetration. Go to Materials and Methods Electro spinning All reagents Sigma unless noted. Polycaprolactone (PCL: 65,000 M.W.) was dissolved in trifluoroethanol (TFE; 150 or 250mg/mL) and placed into a syringe. Syringes were capped with an 18-guage blunt-tip needle and placed into a syringe driver (Fisher Scientific) programmed to deliver a total volume of 3mL of solution at a constant rate of 9mL/hr. Spell men, low current, high voltage power supplies were used to generate electric fields (17-19kV) across a 20cm air gap. A rotating (700rpm) and translating (4cm/s over a 12cm distance) mandrel (6mm diameter) was used as a target. After electro spinning was complete scaffolds were cut longitudinally from the mandrel and stored in a desiccators until needed. Air impedance electro spinning Conditions used to electro spun conventional scaffolds were replicated with the exception that a hollow perforated stainless steel mandrel equipped for air impedance was used as a target to collect the scaffolds [11]. The collection mandrel (6mm in diameter) was engineered with 0.75mm diameter circular perforations spaced every 2.0mm circumferentially (center-to- center) around the mandrel and every 1.5mm down the long axis (center-to-center) of the mandrel. Air flow was introduced into the mandrel at pressures of 0kPa (static), 100kPa, and 300kPa. Computational fluid dynamics (CFD) All computer drawings and meshes were generated using Gambit (Version 2.4). Fluid model simulations were performed in Fluent (Version 12.0) using 1,000 iterations or until convergence was achieved. Graphical representation of the fluid models was visualized using Tec plot [10]. Cell culture Human dermal fibroblasts (hDF, Cascade Biologics C-013- 5C) were cultured in DMEM-F12 (Gibco) plus 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (P/S, Invitrogen. Electro spun scaffolds punch cut into 6mm diameter circles were sanitized in 70% ethanol for 30min, rinsed 2X in phosphate buffer saline (PBS) and transferred to serum free media. For cell seeding (2 5,000 cells) a sterile glass cloning ring was placed onto the scaffolds. Cloning rings were removed after 24hr; cells were cultured at 37°C and 5% CO2. Media changed every 3 days. To explore how passive processes govern cell penetration into electro spun scaffolds cells were suspended in PBS supplemented with 5% para formaldehyde for 30 minutes. The dead cells were rinsed in PBS supplemented with BSA to block any reactive groups prior to plating onto electro spun scaffolds. Cultures plated into electro spun scaffolds also were exposed to daily 15 minute incubations in trypsin (0.025%) prepared in PBS daily beginning on day 1 after plating for 5 days and isolated for analysis on day 7. Control cultures were exposed to a 15 incubation in PBS+EDTA in the absence of trypsin. After each incubation cultures were rinsed and returned to media. Cryo sectioning Scaffolds were fixed 10min in 10% glutaraldehyde, rinsed in PBS and transferred to 30% sucrose solution in PBS for three days at 4°C. Samples were infiltrated with Optical Temperature Cutting compound (OTC), frozen and cut en face into 50|im thick sections. Cut samples were placed immediately in to PBS and stored at 4°C. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).This approach provides a more comprehensive analysis of cell distribution than conventional cross sectional analysis (sections taken perpendicular to the surface of the scaffold) and allows us to reconstruct the distribution of cells in 3D [12]. Image analysis and 3D reconstruction All images were captured on a Nikon TE300 microscope using a 10x or 20xobjective using a DXM 1200 digital camera, images were collected at a resolution of 1280x1024. A bright field image was captured in conjunction with each corresponding fluorescence image of the DAPI stained nuclei. Bright field images were overlaid with corresponding fluorescence images in Adobe and combined using the photo merge function. Individual optical images from each frozenen face section were assembled using fiduciary marks captured in the bright field image into a montage of the entire 6mm diameter, 50um thick frozen sections. This process was repeated for each 50um thick section containing cells, once the entire depth of the scaffold was imaged and reconstructed as a montage the bright field images of the scaffold were removed using a threshold filter. Montage images were imported into Google Sketch Up for 3D reconstruction. DAPI stained nuclei were counted and then converted into cylinders for visualization in the 3D reconstruction. Full details see Grey and [12]. Statistics All data sets were analyzed in Sigma Plot and screened using ANOVA. The Holm-Sidak method was used for pair wise comparisons. P values as provided. Graphical depictions represent±the standard error unless otherwise noted. Go to Results Air impedance electro spinning Computational Fluid Dynamics Air impedance electro spinning was designed to produce scaffolds exhibiting systematically placed macro-pores by forcing air through openings engineered into the target mandrel. Fiber deposition is altered in the vicinity of the openings in the mandrel, presumably by the outflow of air through the mandrel ports. This results in the formation of macro pores that are designed to facilitate cell penetration by providing regional domains of decreased fiber density. We note, in the absence of air flow regional variations in fiber density are still observed in conjunction with the underlying ports of the mandrel, suggesting that air flow and electrostatic edge effects combine to alter fiber deposition in these domains (Figure 1). Click here to view Large Figure 1 Computational Fluid Dynamics (CFD) was used to explore the assumptions that underlie air impedance electro spinning. Theoretically, it is necessary to produce an even outward flow of air through the pores of a ventilated mandrel in order to produce periodic macro pores that exhibit similar structural properties. CFD modeling experiments demonstrate this condition is difficult to achieve, and given the apparent electrostatic edge effects associated with the engineered mandrel ports, may not be necessary. At an inlet pressure of 100k Pa CFD predicts that flow is very heterogeneous along the length of our model ventilated mandrel. Figure 2 offers a convenient visualization of air velocity with respect to pore location in a ventilated mandrel. In this example the mandrel exhibits 120 pores spaced apart at 1.5mm internals (pore center to center) along its length, air is introduced at the proximal end of the mandrel and the distal end of the mandrel is sealed. Both the color and length of the vectors presented in this figure are correlated to velocity (e.g. highest exit velocities are depicted in “long” red bars). Especially notable is the prominent distal velocity bulge that represents increased air velocities predicted at the pore regions at the end of the mandrel farthest away from the inlet air source. The bulk of the air travels down the length of the mandrel and preferentially exits pores located at the distal end of the mandrel. In practical use this disparity of flow is readily detectable by touch when such a mandrel is in use. The introduction of various internal geometries into the mandrel proved largely unsuccessful at altering the preferentially exit of air from the distal pores. The most effective modification to the mandrel design was simply reducing the total length of the mandrel. Through this approach we were able to reduce the mass flow rate (MFR in (kg/s) differential (most proximal pore vs. most distal pore) from 215% in the 120 pore model down to 99% by reducing the "pore length” of the mandrel to 60 pores and finally down to 20% by reducing the "pore length” to 30 pores (while maintaining the same pore spacing-data not shown). While these results show that differences in air flow across the length of the mandrel can be reduced, there were no obvious simple design modifications that could be implemented to even out the flow through the pores. To account for these properties we chose to use only electro spun material deposited onto the proximal pore regions of a mandrel exhibiting a 120 pore length. While these regions displayed the least amount of air flow in our CFD models the pores in these domains exhibited the most uniform flow. Click here to view Large Figure 2 To justify this approach, we compared the MFR (kg/s) of the air flowing through each pore down the length of the mandrel. Based on repeated CFD (N=3) runs we determined that the most proximal 75% of the scaffold (pores 1-41) exhibited air flow rates that were not significantly different from one another and therefore should produce a scaffold exhibiting consistent physical properties (Figure 3). By comparison, the remaining distal pores (pores 42-52) exhibited significantly higher air flow rates and do not represent a consistent fabrication environment (Figure 4). The MFR in the distal pores varied so abruptly (pore 52 displayed significantly higher flow rates compared to pores as close as pore 48, p=. 009) that any test samples taken directly adjacent to each in this region other would have been fabricated under very different conditions. Click here to view Large Figure 3 Click here to view Large Figure 4 Cell culture experiments We cultured human dermal fibroblasts (HDF) for varying intervals of time on scaffolds produced using a solid mandrel ("conventional electro spinning”), a perforated mandrel subjected to an inlet pressure of 100kPa and a perforated mandrel with no air flow. For constructs produced on the ventilated mandrels we only collected scaffolds that were recovered and prepared for cell culture from the proximal regions of the mandrel where CFD predicted air flow to be the most uniform. We made no effort to set a minimal threshold number of cells to count inside the scaffolds; rather we plated an equal number of cells on each scaffold and determined the maximal depth reached on each scaffold. Plotting the maximum cellular infiltration depths vs electro spinning conditions as a function of time revealed very little difference across the treatment groups (Figure 5). Surprisingly, scaffolds collected onto a perforated mandrel in the absence of air flow appeared to support cell infiltration to approximately the same degree as those scaffolds collected with air flow. Click here to view Large Figure 5 After day one of culture, cells plated onto a conventional scaffold were limited to the first 50um of the scaffold. In contrast cells were found as deep as 200um in the scaffolds prepared on the perforated mandrels in the presence and absence of air flow (Figure 5). It is clear from these results that electrostatic edge effects associated with perforations in the electro spinning mandrel likely disturb the fiber deposition nearly as much as the flow of air. Cells penetrated to an equal degree during the initial states of culture in scaffolds fabricated with and without air flow. After the initial 24hr incubation there was virtually no further penetration into the scaffolds prepared on the perforated mandrels over the next 14 days. To penetrate a scaffold by active migration events to a depth of 200um over a 24hr interval would require a polarized and sustained migration velocity along a straight trajectory of approximately 8um per hour (200um of depth/24hrs), this despite the tortuous nature of the interconnected pores of an electro spun matrix. This velocity is possible and well within the capacity of many types of interstitial cells in vitro on a 2D surface [13], however it seems untenable that cells would sustain migration at such a velocity along a linear polarized tract in the environment afforded by an electro spun matrix. Critically, two questions must be asked of these results. First, if cells can reach a depth of 200um over the first 24hr by an active migration process why the deeper regions of the scaffolds are so sparsely populated after 7 days? and, secondly, why should cells on the dorsal plating surface cease further movement after 24hr and not continue to penetrate into the scaffold as a function of time in culture? Our results suggest that variables beyond active migration govern cell penetration in these scaffolds. It is clear that the macro pores must play a role in the process; perceived cell density is highest in regions immediately subjacent to these regions (Figure 4). To investigate the role of passive processes in the penetration of cells into air impedance scaffolds we incubated cells in par formaldehyde and then plated the dead cells for 24hr. At the conclusion of the "plating” interval dead cells were found to be present as deep as 300um into the scaffolds (Figure 6). Click here to view Large Figure 6 These data demonstrates that cells passively sieve into the spaces afforded by an electro spun matrix and become lodged within the pore spaces when the volume of those spaces limits further penetration. In conventional tissue culture live cells will attach to a variety of surfaces through non-specific electrostatic interactions, spread out and begin to express elements of the extracellular matrix. These events must largely be reproduced within the 3D environment of an electro spun matrix and likely greatly suppress cell penetration [14]. If cells that have established early contacts with the fibers of the matrix could be systematically dislodged from those surfaces penetration into deeper regions of the scaffold might be achieved. To explore this hypothesis we examined how repeated exposure to trypsin might disrupt early cell matrix interactions and promote cell penetration into electro spun constructs. Trypsin digest is the classical method used to disrupt cell matrix adhesions in tissue culture. To explore the utility of this method with cells trapped within an electro spun matrix we plated cells onto electro spun scaffolds and exposed them to daily 15min incubation in trypsin. In these experiments we utilized conventional scaffolds electrospunonto a solid mandrel using 250mg/ml PCL solutions. It is clear from cell counting data, (Figure 7 & 8), cross sectional analysis and 3D reconstructions (Figure 9) that cyclic trypsin treatments can promote increased cell penetration into electro spun scaffolds. Click here to view Large Figure 7 Click here to view Large Figure 8 Click here to view Large Figure 9 Go to Discussion These data indicate that passive processes largely govern the penetration of cells into an electro spun scaffold composed of a synthetic polymer. Cells sieve into the interconnected pores across the Z axis over the first 24hr. This conclusion is supported by two lines of experimental evidence. First, a sub-population of cells rapidly populate the deeper recesses of an electro spun scaffold over the first 24hr of culture, subsequent penetration is nearly non-existent and a large population of cells remains on the dorsal plating surfaces of the scaffolds. Second, even par formaldehyde treated cells are observed to penetrate into the scaffolds. The lack of physical guidance cues likely limits the contributions of active migration in the penetration of cells across the Z axis of these constructs. The active events of cell migration, when they take place, appear to predominately occur in parallel with the fiber layers rather than across the fiber layers. Air impedance electro spinning was developed to promote the infiltration of cells across the Z axis of an electro spun scaffold [11]. The introduction of systematically placed regions of lower fiber density produced by air flow through a ventilated mandrel is intended to increase the porosity of a scaffold without substantially compromising its material properties. Our data (Figure 5) demonstrate that electrostatic edge effects play a central role in producing macro pores in these scaffolds. Given these results it would appear that very little advantage is to be gained by impressing air flow into this system, CFD modeling reveals that achieving an equal air flow-even in the absence of fiber deposition- is difficult to achieve, even on a small scale. However, we recognize that air effects may have more pronounced effects on modulating fiber density on thicker constructs. The decreased fiber density associated with the macro pores fosters increased cell penetration (Figure 4), albeit this penetration appears to be primarily by passive sieving of cells into the scaffolds. We note that scaffolds fabricated with native proteins may be less likely to support sieving as well as a purely synthetic scaffold. The cell specific adhesion sites of such matrix would provide far more stable cell-matrix contacts than the nonspecific electrostatic interactions that govern the early stages of contact between a cell and a synthetic biomaterial. Constituents of the native extracellular matrix are arrayed in a many different orientations (X,Y and Z), in contrast, the fibers of an electro spun scaffold are deposited as a series of discreet layers in which the longitudinal axis of the fibers lies parallel to the target mandrel. This structural characteristic provides very large pore spaces between the fiber layers along the X-Y axis, a characteristic that provides very little impediment to movement along this axis (parallel to the dorsal surface). Previous experiments form our laboratory suggest that cells deep in a scaffold along the periphery of the construct largely originate with cells that spill over the edge of the construct during cell plating and have entered the scaffold along its lateral borders [12]. We have explored this consideration by producing cylindrical electro spun nerve guides composed of linear, parallel arrays of fibers [15]. The "enhanced porosity” or perhaps the lack of physical barriers present between the longitudinally arrayed fibers in combination with the guidance cues provided by the polarized fiber tracks in these devices provide a potent mix of signals to promote directed cell migration and axon elongation in vitro and in vivo [10,16]. The pores spaces present in an electro spun scaffold along the Z direction, while extensively interconnected, are far smaller and vary considerably in geometry. In this orientation spatial considerations will limit the extent to which cells can travel along any particular axis [9]; this is one reason why we find active migration to be untenable explanation for the extent to which cells penetrate our scaffolds over the first 24hr of culture. Any migration across the fiber axis and into the scaffold would necessarily be highly tortuous and cover far more distance than the linear measured distance between adjacent tissue sections. There is no clear way to fabricate an electro spun matrix that more closely mimics the heterogeneity of fiber polarity that exists in vivo. It does not seem likely that scaffolds composed of fibers traveling in the X-Y axis and Z axis can co-exist in the same electro spun structure. We have attempted to provide a mechanical gradient by fabricating scaffolds composed of fibers that vary in diameter as a function of the Z direction in a scaffold [10]. These constructs are highly resistant to delamination failures, but do not appear to promote any more extensive cell penetration than conventional scaffolds. We believe the material properties of the individual fibers that make up the mechanical gradient are "out of range” for the cells to actually detect (fibers are too stiff). We were successful in this study at promoting the infiltration of the scaffolds using a cyclic trypsin digest. This treatment would be expected to disrupt any cell-matrix interactions formed as a result of fibronectin, vitronectin, other serum constituents and or cellular activity. The loss of these adhesions will cause the cells to round-up and perhaps makes them more susceptible to sieving into the scaffolds. The impact of this treatment on increasing cell division also cannot be discounted as a mode by which the enzyme functions to increase cell number inside the scaffolds [17-21]. If this type of intervention proves effective for other cell types it may be possible to populate scaffolds with a broad spectrum of cell types relatively easily and quickly, including differentiated cells that are ordinarily relatively immobile, like muscle cells. Ultimately, diffusion barriers will limit the extent to which tissue engineered materials can be populated in vitro. However, in order to successfully produce engineered tissues and organs the limits of that barrier must be fully explored and defined. Developing strategies to bump up against this barrier are critical to the future success of the tissue engineering paradigm. For more articles in Open Access Journal of Material Science please click on: https://juniperpublishers.com/jojms/

#Juniper Publishers #material science #composite materials

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