Diabetes Research- Which Metabolic Processes to Target?

Diabetes is a global concern for the healthcare community. It is a chronic metabolic disorder that can either be Type1 (failure to produce enough insulin) or Type2 (inability of the body to use insulin). The exact cause of Type1 Diabetes is still under research and is believed to be an autoimmune disorder with the involvement of genetic and environmental factors. Type2 Diabetes is generally caused by poor lifestyle and diet habits. In the case of untreated Diabetes, blood sugar increases in the blood, leading to hyperglycemia and organ damage.

With an increase in diabetes prevalence, the global research effort to find diabetes treatment is increasing and the market is predicted to expand from $50 to $80 billion (2018-2026). In this article, we have discussed some commonly used in Vitro assays to study diabetes using cellular models and biochemical platforms.

Commonly Targeted Metabolic Pathways for Diabetes Research

diabetes study
Image source: PromoCell

Here are some common metabolic pathways or processes in the Diabetes study. Studying these pathways can help in finding new targets and drugs for Diabetes treatment.

  • Lipogenesis

The presence of insulin signals the cells that glucose is abundant and can be stored as fat. This generates triglycerides in hepatocytes and adipocytes. The triglycerides can be measured for diabetes studies.

  • Lipolysis

Instead of breaking down fats for energy, cells use glucose when insulin levels are high. This breakdown of triglycerides is lipolysis. This process breaks down triglycerides into glycerol and fatty acids. Glycerol assays can help in measuring this pathway product.

  • Gluconeogenesis

Glucose assays can be used to determine glucose levels. In case insulin is present, new glucose molecule production through gluconeogenesis is inhibited.

  • Glucoseuptakerate

Glucose uptake assays can measure the rate of uptake by the cells. Insulin helps in speeding up glucose uptake by glucose transporters.

  • GLUT4 translocation

In presence of insulin, GLUT4 transporters are translocated to the plasma membrane for uptake of glucose. The measure of GLUT4 translocation is possible using TRF (time-resolved fluorescence) assay.

  • Glycogenesis

In presence of insulin, the storage of glucose is upregulated in myocytes and hepatocytes in the form of glycogen. Glycogen levels can be measured by glycogen assay.

As more people are succumbing to Diabetes, more research assays to study Diabetes are coming up. Metabolomics is becoming a crucial domain of support in understanding the metabolic pathways to detect and study Diabetes. The metabolic processes mentioned above have been commonly used by researchers and clinicians to study and diagnose Diabetes. With the advent of better-advanced technology, these assays are getting technologically modified platforms in the form of 3D cell culture and microfluidics. Diabetes research is progressing towards excellent pharmaceutical and metabolic discoveries and KOSHEEKA is proud to be a part of this progress with its range of tissue-specific primary cells for academic and industrial research. Contact info@kosheeka.com for more info on Diabetes research and models.

Animal Cell Culture- Safety and Handling Considerations

A crucial step before taking up research work with human or animal tissue is to ensure appropriate ethical and medical legislation and guidelines for experiments. This can also deal with procuring approval from relevant authorities or individuals.

Biohazards and Safety considerations

It is very important to know the possible associated risks while working with potential biohazards. This happens only with a sound knowledge of the materials and working protocols. Cell cultures are generally considered biohazard prone as they can easily harbor infectious agents like viruses.

Biohazard degree depends on the cells in the culture and the experiment to be performed. Primary cell cultures need very careful handling as they have higher risks of getting contaminated with undetected viruses or mycoplasma. Cell lines should also go through proper screening before being used for experiments as contaminated cultures will adversely affect the research results.

Every animal cell culture lab should maintain proper documentation on handling cell culture work to avoid any risk of potential infection of the environment. Good laboratory practices are essential for two main reasons: (a)reducing the risk of exposure for the workers, and (b)preventing cell culture contamination with microbial or other cells. Working in a biosafety-approved laminar flow hood requires researchers to follow stringent aseptic techniques, ensuring aerosol limitations. aerosols represent an inhalation hazard, and can potentially lead to cross-contamination between cultures. To avoid aerosol formation, TD (to deliver) pipets should be used instead of TC (to contain) pipets. Moreover, some more tips to avoid aerosols are:(a) using pipets with cotton plugging,(b) not mixing liquids by rapidly pipetting, (c) not using excessive force while using pipets, (d) not bubbling air through liquids while using a pipet, (e) releasing the contents of a pipet as close as possible to the liquid level in the vial or allowing the contents to run down the vial sides. Besides these simple tips, proper usage of equipment, like a centrifuge, can also help in minimizing the risk of aerosol formation. After laminar work, all the waste media and consumables should be disinfected using autoclave or disinfectants before proper disposal according to institutional or organizational guidelines.

Five Simple Lab Tips to Use Laminar Hood

  • Keep laminar flow hoods in an area with minimal air current disturbance. Avoid placing them near doorways or air vents. Have dedicated sterile cell culture rooms for your cell handling.
  • Laminar flow hoods should be kept clean without storing equipment inside the cell culture hood.
  • Before starting work in the laminar hood, surface disinfection of the laminar stage, bottles, etc. should be done using 70% ethanol or IPA.
  • Arrangement of pipets, waste containers, reagent bottles, etc. should be done in a way that one can avoid passing used items over clean items or open culture dishes or flasks.
  • Used items for discard should be kept in a bin inside the hood till all work has been completed. Before removing the discard bin, disinfect with ethanol.

If your lab is involved in primary cell culture or stem cell culture research, connect with KOSHEEKA at info@kosheeka.com for the best cell culture solutions.

5 Things to Know about Primary Cells

Primary cells mimic the tissue of origin as they are directly isolated from the tissue and processed for culturing under optimized media conditions. Therefore, primary cell culture provides excellent model systems for studying normal cell physiology and biochemistry. There are a lot of articles on primary cells vs cell lines to understand which one of them enhances research efficacy but in the case of primary cells (take for example, drug studies), the research results are closer to the effects of drugs on normal body physiology. Primary cell culture researchers work with different types of primary cells based on tissue-specific, species-specific, and disease-specific categories as per their research requirements. But to understand primary cell culture better, here are five things to know about primary cells.

* Growth Requirements

Primary cells can be grown in suspension or adherent cultures. Some primary cell examples cells like peripheral blood cells naturally grow in suspension, without surface attachment. These primary cells grow to a higher density than the possible limit of adherent conditions. For the types of primary cells that are anchorage-dependent, these adherent cells require a surface to grow in vitro properly. Adherent primary cells are mostly cultured in a plastic vessel, but they can also be cultured on a micro-carrier (coated with extracellular matrix proteins to increase adhesion and provide growth and differentiation signals. The cell culture media for primary cells is composed of a basal medium with growth factors and cytokine supplements. Primary cells are grown and maintained at typically 37 °C, 5% CO2 (for mammalian cells) in a cell incubator. The culture conditions widely vary depending up on the types of primary cells. Primary cell growth media can vary in pH, glucose concentration, growth factors, and nutrient composition, depending up on the primary cell types.

During the establishment of primary cell cultures, it is essential to include antibiotics (may include a mixture of gentamicin, penicillin, streptomycin and amphotericin B) in the growth medium for limiting contamination from the host tissue. However, the long-term use of antibiotics is not recommended, as some reagents may be cytotoxic or cause problems during secretome analysis.

Retaining the viability of primary cells after isolation is crucial as they undergo senescence and stop dividing after a certain number of passages. For long-term viability of the primary cells, excellent primary cell culture handling skills along with aseptic culture techniques and appropriate culture conditions are essential.

* Cellular confluence

Primary cell confluence generally refers to the percentage of the cell culture flask or dish that the cells inhabit. A 100% primary cell confluence means that the surface area of the culture vessel is completely covered by cells, whereas 50% primary cell confluence means half of the surface is covered. Primary cells are never grown to 100% confluency as the chances of senescence increases and leads to increased cell loss. The limit of confluence is a major factor that determines subculturing period and cell health after subculturing.

* Maintenance and Subculture

The primary cell culture maintenance phase begins after the attachment of the isolated cells to the dish or flask surface. Usually, cell attachment takes about 12-24 hours after initiation of the primary culture. When cells reach a desired confluence and are actively proliferating, subculturing process is the next step. Never let the cells reach a 100% confluency as post-confluent cells may undergo differentiation and exhibit slower proliferation.

Adherent primary cells grow in monolayers and needs sub-culturing at regular intervals with appropriate culture medium for maintaining exponential growth. Sub-cultivation involves the breakage of both inter-cellular and intra-cellular bonds using proteolytic enzymes like trypsin/EDTA. After cell-attachment dissociation and single-cell suspension, the primary cells are counted and diluted to appropriate concentration before transferring into fresh culture vessels for growing.

* Cell counting

Hemocytometers are generally used for primary cell count and cell viability determination using the exclusion dye Trypan Blue. For information on cell counting, check out https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-quantification.html . In case of primary cells, although the method for counting cells is simple and considered a universal gold standard, it is difficult to achieve consistency among lab researchers as it becomes subjective based on handling of the subculturing process.

* Cryopreservation and recovery

In case of primary cells, cryopreservation is achieved using a cryoprotectant, such as DMSO or glycerol. Generally, cryopreservation can be achieved in 80% complete growth medium supplemented with 10% FBS and 10% DMSO. The freezing has to be slow, at a rate of -1°C per minute, to minimize ice crystal formation within the cells. Finally, the sample needs to be stored in the vapor phase of liquid nitrogen (-196°C). Precaution should be taken to avoid centrifuging the primary cells right after thawing as they are extremely sensitive to damage during the recovery phase.

For more information on primary cell culture, contact Kosheeka at info@kosheeka.com with your primary cells and stem cells inquiries.

Applications of Primary Cell Culture in Research

Cell culture is a prominent tool in cellular and molecular biology research, often spreading to clinical applications. Cell culture using primary cells can be excellent model systems for studying physiology and biochemistry of cells, drug toxicity and metabolism, and other biomedical applications. Primary cell culture is being used by researchers worldwide for their role in maintaining the consistency, efficiency, and reproducibility of results, besides being an excellent mimic model of in vivo physiological conditions. The applications of primary cell culture can be termed as:

  • 3D Model System

Primary cell culture can be used as 3D model system to study cell biology and biochemistry, cell interactions, pathological interactions, drug effects and toxicology, aging and several other research domains.

  • Cancer Research

Difference between normal cell and cancer cell can be studied using primary cell culture as screening of over-expressed or under-expressed markers leading to oncogenic activation can be detected using primary cell culture as model. Moreover, using primary cells as control models for anti-cancer research is significantly exploited.

  • Virology

Primary cell cultures are used to replicate viruses and can also be used to detect and isolate viruses for studying their growth and development cycle. Primary cells are also used in virology to study the mode and mechanism of virus infection.

  • Toxicity Testing

Primary cell culture is used to study the effects of new drugs, cosmetics and chemicals on cells. Primary cells are also used to determine the maximum permissible dosage of the drugs.

  • Vaccine Production

Cultured primary cells are used in replication of viruses for producing vaccines. Vaccines for polio, rabies, chicken pox, and hepatitis B are produced using primary cell culture.

  • Genetically Engineered Protein

Primary cell cultures are used to produce genetically engineered proteins such as monoclonal antibodies, insulin, hormones, etc. Such recombinant technology is utilized in reshaping many therapeutics.

  • Replacement Tissue or Organ

primary cell culture can be used as replacement tissue or organs in wound healing and other tissue engineering applications. E.g.- artificial skin can be produced using primary cells to treat patients with burns and ulcers. Research is on-going for artificial organ culture such as liver, kidney and pancreas. In this regard many researchers are taking up stem cell culture as these cells have the potential to self-proliferate and differentiate into several cell lineages. Many therapeutic applications of adult stem cells are currently in trials for tissue / organ regeneration and replacement technology.

  • Drug Screening and Development

Primary cell cultures are used to study cytotoxicity and safe dosages of new drugs. Primary cell cultures therefore play an important role in the pharmaceutical industry.

Primary cells have become an indispensable part of today’s efficient biomedical research and if your lab is looking for tissue-specific and species-specific primary cells or stem cells, you can contact Kosheeka at info@kosheeka.com for the best cell procuring experience in India.

Advantages and Disadvantages of Using Primary Cells in Cell Culture

Primary cells  represent the in vivo tissue environment fairly and these cells are directly taken from body tissues for processing and establish them under optimized culture conditions. As these cells are derived directly from native body tissue and not modified, they mimic the in vivo state and physiology. Therefore, they provide excellent model systems for studying physiology and biochemistry of cells involving metabolic studies, signaling studies, drug toxicity etc. The most popular primary cells examples used in research are epithelial cells, fibroblasts, endothelial cells, keratinocytes, melanocytes, muscle cells, hematopoietic, and mesenchymal stem cells. These types pf primary cells are initially heterogeneous and can be maintained only for a limited period of time in vitro. When primary cell cultures undergo genetic transformation, they divide indefinitely and become immortalized secondary cell lines.

Primary Cells vs Cell Lines

Continuous types of cell lines  have the ability to proliferate indefinitely, are generally more robust, and easier to work with than primary cells. A major drawback of working with continuous cell lines: they are genetically modified and that can alter physiological properties in case of extensive passaging. A table for Primary cells vs cell lines has been given below for clear understanding.

Primary Cells vs Cell Lines


Advantages and Disadvantages of Primary Cells

Advantages of Primary cells:

* Bypasses ethical objections against animal use in biomedical research and allows human tissue experimentation.
* The use of primary cells provides more relevancy to biomedical research than using cell lines. Pre-screened primary cells can be good systems to study biochemical signaling vivo very closely.
* Primary cells reduce the expenditure on animal models and in turn make research cost—effective.

Disadvantages of Primary cells:

* Primary cells take more growth time than cell lines and have limited growth potential. Even with optimal growth conditions, primary cells eventually senesce and die without going for more passages.
* The cells taken from different donors might behave differently in case of immune responses (unless pre-screened).
* The cost of primary cell isolation and culturing is often high and primary cell cultures mostly require good amount of handling expertise. Primary cell characteristics may change with each subsequent passage in case optimum culture conditions are not maintained.
Although primary cells are relevant to biomedical research and give a boost to ethical research practices, procuring primary cells is a challenge. Availability of tissue-specific and species-specific primary cells is a basic limitation for many labs in India and this is where Kosheeka comes to help biomedical research ventures. Kosheeka provides the best quality of primary cells, customized as per your lab requirements. Check out our cell listings at http://kosheeka.com/ and mail us your inquiries at info@kosheeka.com

A Brief Insight On Mammary Epithelial Cells And Carcinoma

The growth of normal human mammary epithelial cells, including luminal, myoepithelial, and/or basal cells, is tightly controlled. Mammary epithelial cells grow for a finite span and eventually die or undergo senesce. Human, rat, and murine mammary epithelial cell have provided evidence that essential initial steps in mammary carcinoma cell line growth involve the loss of senescence checkpoints for encouraging immortalization. In addition, mammary epithelial cell culture model systems have identified a number of genes whose alterations are involved in mammary carcinoma cell line development. Additional insights come from using transgenic overexpression of carcinoma-promoting genes or deletion of cancer suppressor genes. Let us get some insights into mammary epithelial cells and their cancer progression.

Mammary Epithelial Cells

The mammary gland consists of a branching ductal system that ends in terminal ducts with their associated acinar structures (terminal ductal-lobular units or TDLUs), along with interlobular fat and fibrous tissue. Histological examination of the TDLU has shown two major types of cells: inner secretory luminal cells and outer contractile myoepithelial cells. Two types of luminal cells are present lining the mammary gland ducts and alveoli. In addition to these, there is also evidence regarding the presence of stem cells and progenitor cells for mammary epithelial cells. 

For more than two decades, researchers have attempted to develop mammary epithelial cell culture models that resemble human breast cancers in vivo. In order to establish such models, culturing non-cancerous mammary epithelial cells was necessary using a human mammary epithelial cell growth medium. To design an optimum growth medium, researchers prepared a defined medium DFCI-1 to culture mammary epithelial cell and epithelial carcinoma cell lines but there was difficulty in establishing primary carcinoma cell culture.

Mammary Epithelial Cells and Carcinoma

Cultures derived from reduction mammoplasty or mastectomy specimens exhibit considerable heterogeneity but researchers devised ways to establish mammary epithelial cell from these specimens. In the procedure, the tissue is finely chopped, digested by collagenase and hyaluronidase, and plated as organoids. Over a week, multiple types of epithelial cells and fibroblasts are seen but fibroblasts are removed by differential trypsinization, leaving mammary epithelial cells. For information on mammary epithelial cell growth medium or serum-free cell culture media, click here 

Mammary Epithelial Cell Carcinoma

Mammary carcinoma exhibit both inter-and intra-tumoral heterogeneous nature. Several study reports have indicated that human mammary epithelial cell cancers exhibit diverse phenotypes according to pathological features and therapy response. Studies have identified distinct gene profiles to classify mammary carcinoma cell lines. Five categories of mammary carcinoma cells can be described as a basal epithelial-like group, ErbB2-overexpressing group, normal mammary epithelial cell-like group, luminal epithelial cell type A, and luminal epithelial cell type B. Importantly, these molecular classifications provide a strong rationale for studying various mammary epithelial cell subtypes and models to understand carcinoma cell line molecular diversity.

Further gene expression profiling shows that within each subtype, tumors can exhibit further variability in gene expression and drug susceptibility, making sense of distinct patient complications. Molecular profiling studies report that the gene expression patterns of cancer subtypes align with normal mammary epithelial cell lineages and this suggests that tumor subtypes may originate from distinct mammary epithelial cell subpopulations. It is widely believed that mammary epithelial stem cells/progenitor cell populations may serve as carcinoma initiating cells since their longevity and self-renewal ability can afford genetic mutation accumulation. For more information on mammary epithelial cell carcinoma and its hierarchy, click here

If your lab is working on mammary epithelial cell carcinoma, Kosheeka can help you procure the best quality of human primary cell culture, tissue-specific primary cells, and disease-specific primary cells. Contact info@kosheeka.com for further inquiries.

Importance Of Basal Media And Supplements In Primary Cell Culture

The task of choosing cell culture media for use with your primary cells is surely daunting. Therefore, if you are thinking of replicating a formulation of media from any previously published report or if you are optimizing your nutrient or classical media, it is important to understand the consequences of this media choice for your primary cell culture maintenance and research.

Many biological researchers who work with cell lines, commonly use nutrient and classical media as they deem sufficient for cell line growth and maintenance. But Primary Cell culture requires additional nutrients for enhanced culture practice. Cell lines are more proliferative in nature with a better adaptation to 2D culture and they tend to be more resilient to cultural conditions. In the case of primary cells, they are isolated from their 3D host tissue and then transferred to a 2D environment of dish or flask.

These cells are less proliferative than cell lines and have more complex requirements for nutrition. Primary cells also have a limited lifespan and reach towards senescence faster in culture conditions. Due to these special features, special media is a better choice for primary cell cultures and this calls for optimized basal media with supplementation. Specialty media are designed to be used with little or no serum component and supplemented with growth factors, lipids, and hormones. These components allow the proper optimization for growing the specific cell type.

Researchers have done some studies to choose the correct form of basal media and supplementation in form of culturing primary cells with company-composed specialty media and DMEM/F12, as this is a good supportive basal medium, for comparing the growth and maintenance of primary cells. Moreover, some studies also compared this medium with higher serum levels (10%) versus lower serum levels (5%) in combination with endothelial cell growth supplements, hormones, and growth factors for the optimized growth of endothelial cells. Studies showed that primary cells grew more quickly with the formulation having 5% serum as compared to 10%, thus suggesting that a lower serum formulation, in combination with growth factors and hormones, is better for primary endothelial cell proliferation.

But better than DMEM/F12 and other supportive basal media, the basal media closest to the extracellular matrix (ECM) environment showed enhanced growth and better compact morphology. Thus, the correct composition of specialized basal medium with growth factors and supplements, when it mimics the ECM of specific primary cells, is important to understand for having the optimum proliferation and viability of primary cells in culture. Always remember, the healthier your primary cells stay, the more relevant your results are!

At Kosheeka, we consider the complex nutrition needs of the primary cells and help you choose the best media and supplements for your cell culture. With our cell culture expertise, information, and resources, procuring primary cells, customized to your research needs, is quite an easy and affordable feat, and for any inquiries related to primary cell culture and Stem Cell Culture, contact us at info@kosheeka.com

Simple Lab Tips For Primary Cell Culture

Primary cell culture is an expanding technology for several biomedical applications like tissue culture, 3D Cell Culture, bioscaffolds, 3D bioprinting, organoid culture etc. Researchers have been utilizing this technology as an efficient ethical alternative to animal experiments and as a physiologically in vivo-mimicking alternative to culturing continuous cell lines. We, at Kosheeka, believe that cell culture practice and maintenance can enhance the applications and efficacy of primary cell culture manifold and therefore, here are some simple tips for Primary Cell Culture maintenance that will hopefully help the budding researchers.

Maintain Your Hood

The biosafety cabinet should be turned on for at least 15 minutes before the work in the hood starts. This is to ensure proper clean air flow. The air flow vent opening should be left uncovered to ensure proper flow. Moreover, before work starts, a 10-15 minutes of switching on the UV light ensures that no contamination in the hood stays. UV light is harmful for your eyes and skin and therefore, keep it off while using the hood. After switching off the UV, always wipe the hood surface clean with 70% ethanol or IPA before starting work.

Keep Your Contaminants Away

Like we suggested in the previous point, wipe down your working surface of the hood with 70% IPA or ethanol and also wipe everything that gets inside the hood, including your glove-wearing hands. Remove rings and other ornaments on your body before working in a cell culture lab. Also avoid talking unnecessarily or touching your skin or hair while inside the culture room. All these may sound cliché and trivial but every small step helps in reducing the chance of bringing contaminants from outside in the form of aerosols or microbial contaminants. Avoid moving things in and out of the hood while working on your cell culture and always plan your experiments ahead.

Keep The Hood Tidy And Clean

Hoods are not for storage! Cluttering your hood for work increases contamination risks and makes it more difficult for air flow and working ease by reducing working area and making it difficult for you to clean the hood surface. Just take what you need inside the hood area by planning it beforehand and keep your tubes and bottles closed when not in use, while working. If media spills or any spills occur inside the hood, they should be immediately wiped clean with 70% ethanol or IPA with sterile tissue papers and discarded in the small beaker (used as bin inside the hood for pipette tips). Moreover, instead of using the whole bottle of any solution or media, aliquot the necessary amounts in falcon tubes for ease and keep the main bottle closed to avoid any chance of contamination of the stock solution. 

Care For Your Cells In The Hood

Do not let your cell culture flasks stay for too long outside the incubator. pH maintenance is a big issue without incubator and growth is affected due to disturbance of the preferred temperature conditions. Primary cell cultures should be handles with utmost care and without a sterile incubator, conditions might be harmful for the cell viability. Keep the cells inside the incubator while you are preparing other media and solutions so that you can avoid clutter and also help your cells to be happy and healthy. Also ensure that unless specified, temperatures of washing buffer like PBS should be similar to the temperature of the cells to avoid any mechanical harm towards the cells.

Check Cell Morphology And Numbers

A light microscope should be available close to the hood to have the least distance between working on your cells and viewing them. One should also know the proper morphology of the cells that they are working with for ensuring proper maintenance of the cells. This helps in detecting cross-contamination and microbial contamination.

When starting a new primary cell culture, a good practice in maintaining cell numbers is to grow the cells, aliquot them in a number of cryovials, cryopreserve them in liquid nitrogen and use just one of those vials to propagate passages. This ensures that if any issues arise in your cell culture passage, you can always lean back on the original stock of cells.

Hopefully, these simple lab tips for primary cell culture will help you enhance your culture practice in the lab settings and if you are ready to start your primary cell culture, Kosheeka is here to make it easy for you! For procuring species-specific and tissue-specific primary cells, contact us at info@kosheeka.com

Common Errors And Tech Tips For Primary Cell Culture

If you are a cell culture researcher working with primary cell culture, the most difficult thing to accept is watching your cell dishes and flasks getting contaminated or not proliferating even with appropriate incubation at the correct media pH and temperature conditions. Watching those adherent cells not attached to the flask surface and knowing that you have to repeat the whole process of maintaining your cell culture for starting your cell culture-based assays and other experiments, is quite taxing for any researcher.

Not only is this a waste of the time and energy of the researchers involved, but also a lot of money and resources like media and other reagents are wasted in the process and this can be close to a nightmare for a financially tight budget-based research laboratory. Therefore, it is very important to know the tricks and treats of every cell culture process step that are practiced in labs to ensure healthy growth and maintenance of the primary cells. Here in this article, we have thus compiled a list of some very common troubleshooting steps in the primary cell culture process so that your cells stay healthy and growing well.

1. Caring for the Cells after Taking out of Cryopreservation

Most researchers think that trouble related to the processing of the primary cells after cryopreservation lies in the amount of cryopreservation agent (DMSO) left in the media after centrifugation but other issues are quite prevalent too. These other issues include omitting the recommended cell seeding density and the recommended media volume per cryovial or per seeding flask. Moreover, the amount of freezing media in the cryovial is also a considerable issue. DMSO in the freezing media can be diluted by using the proper ratio of media to freezing media in the cryovial before taking the cells for cryopreservation. If seeding and media volume are taken care of, then comes the handling of the cells and proper thawing procedure.

Researchers should follow and optimize their thawing procedure to prevent damage of the cells and this step matters a lot in maintaining the viability of the cryopreserved primary cells. After thawing, centrifugation is important to separate the cells from DMSO and media. Recommended centrifugation protocols should be followed to prevent harsh damage to the cells. After seeding the primary cells post centrifugation, always remember to change the media after cell attachment observed to ensure the removal of any residual DMSO.

2. Using Specific Media for Culturing the Cells

Cells love their media environment and if not fed proper nutrients, Primary Cells can be quite adamant to not proliferate and stay unattached, thus giving a hard time to the researchers. Most cell types have specific media for their optimum growth and researchers should know the required nutrients as primary cells can become conditioned to specific culture media and changing the nutrients can lead to a lack of cell attachment when plating and slow proliferation rate. As for an example, endothelial cells can become dependent on VEGF. Therefore, trying to switch to a VEGF-free media can cause the cells to not adhere or grow very slowly and every cell type should be studied well by the researchers before growing them in labs.

3. Re-Frozen and Late Passage Cells

Primary cells should not be treated as continuous cell lines that can go on for several passages. Primary cells can grow up to finite passage numbers and will senescence in due time. To keep up with good cell viability, primary cells should be used within the specified guaranteed number of doublings to prevent damage. It is always advised to cryopreserve early passage cells in case of storing primary cells as later passage cells may not be able to recover without causing significant damage to those cells. Early freezing of the cells also ensure that once some issue occurs in terms of contamination or proliferation, researchers can always go back and start their cultures from early passage numbers to prevent loss of cell viability. 

4. Subculturing Primary cell Reagents

Primary cells often need lower concentrations of Trypsin/EDTA formulations during culturing and passaging of the cells for regulating optimal proliferation and viability. Higher concentration of trypsin can lead to damage of the primary cells and thus researchers should focus on knowing how to maintain optimum media environment for their cells. Moreover, serum concentrations are also an important factor for optimum cell culture practice and some cultures are better grown in serum free conditions. 

If you are looking for highly viable primary cells, Kosheeka can help you procure tissue-specific and species-specific primary cells. Contact info@kosheeka.com and get customized primary cells to enhance your research efficiency.

Primary Cell Culture Cryopreservation: Q&A with Dr. Sachin Kadam

Cryopreservation is an essential cell culture maintenance activity in biomedical labs to preserve cells for future use. There are various aspects of cryopreservation which need to be learnt and properly performed so as to obtain a good viable population of cells after thawing, in case a culture follows. Keeping these things in mind, we had an expert session with Dr. Sachin Kadam on the Do’s and Don’ts of cryopreservation.

Dr. SachinKadam, with over 13 years of experience in stem cell research, has fueled Kosheeka’s research and development with his esteemed expertise and scientific zeal. In this article, we put forward a short piece on our Q&A session about cryopreservation techniques and ethics.

Which type of threaded cryovials are preferred by cell culture researchers: internal or external?

Some researchers prefer external threaded cryovials so that no outside particles or contaminants get in the vial, thus making it safer for cryopreservation. Whereas, some researchers choose internal threaded vials as they fit better in the canisters and freezer boxes.

DMSO is used as a cryopreserving agent. What alternatives would you suggest for primary cell culture applications?

DMSO is an intracellular cryoprotective agent and many alternatives have been reported for tissue freezing, including non-penetrating cryoprotectants like glucose, sucrose, galactose, or trehalose, and intracellular cryoprotectants like ethylene glycol, propylene glycol, glycerol, formamide, methanol, and butanediol. Researchers generally use freezing media with 10% DMSO and FBS for cell culture applications but the use of polyvinylpyrrolidone (PVP) has been suggested in some literature reports as alternative to DMSO.

Which points in the cryopreservation protocols should be primarily focused on?

There are some main points to focus while going ahead with cryopreservation. Firstly, freezing good quality cells is an important criteria. Researchers should be aware that high viability is not related with high population. Cells should be frozen after being passaged for 2-3 days with less than 90 percent confluency. A population of 2 x 10^6 cells/ml is mostly the typical cryopreservation density. Secondly, using DMSO as a cryoprotecting agent along with FBS or Ficoll to the freezing medium. In this regard, researchers should use fresh reagents for freezing procedure to gain high recovery efficiency. A constant rate of freezing at -1°C/min is suited for cell culture applications. Moreover, keeping cryovials in the vapor phase between -140°C and -180°C reduces the risk of leaky vials or unexpected damage. Last but not the least, cells should be thawed rapidly by placing the cryovials in a 37°C water bath to prevent osmotic shock.

Is there any way to store cryovials if liquid nitrogen availability is an issue?

A temperature of -80°C might be a short-term alternative to store primary cells but with the use of 10% Ficoll 70 to the 10% DMSO in the freezing medium can help in enhancing recovery viability.

We thank Dr. Sachin Kadam for his valuable insights on cryopreservation for primary cell culture and a lot of troubleshooting still awaits for various other crucial points in the process of good cell culture practice. For any information on cryopreservation or primary cell culture practices, consult Kosheeka at info@kosheeka.com