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.

Differences and Advantages :Immortal Cell Lines and Human Primary Cells

When you are starting your cell culture research, the primary requirement is having cells, and to decide whether human primary cells or cell lines will be beneficial, is a major task! If you want a cell culture model that is closer to human in vivo environment, human primary cells are a better choice. Human primary cells offer much efficient research efficacy and publication validation if you are planning to have your research work published to inform the global scientific community. On the other hand, cell lines offer a much easier model to work with and are well established in literature but lack the part of ‘mimicking human physiology’.

Human Primary Cells

Human primary cells are directly isolated from tissues and therefore retain the morphological and functional characteristics of their origin. As we can see from Pastor et al., 2010, original tumor tissue from patients preserves several tumor markers and miRNAs. Cell lines do not retain original physiology or markers.

However, human primary cells do not live forever in passages and undergo senescence. With more passage numbers, primary cells also start showing morphological and functional changes. Therefore, it is always advised to use primary cells of lower passage numbers. Furthermore, primary cells also give an advantage of not using animal models, thus having an ethical upper hand.

Human primary cells come from human donors, donated organs, surgical specimens, post-mortem donors, etc. The source limitation is always there while working with primary cell cultures and therefore major planning is crucial while handling primary cells in case of experiments. Also, primary cell cultures derived from explants often do not make homogeneous cultures and purification of specific cell types are required. Primary cells also require additional nutrition and growth factors as they are more sensitive than cell lines. Read here for primary cell culture applications.

Human Cell Lines

Since the early 20th century, researchers have relied on cell lines to gain insights into cell biology and metabolism. Cell lines or immortal cell lines have become a widely used model in cell culture literature, becoming a known and optimized entity for drug studies, biochemical assays, bioactive production, etc. Researchers are comfortable with cell lines as they are cost-effective, work-friendly, and can run for more passages than primary cells. Cell lines are easy to manipulate and expand, making it preferable for multiple screening owing to an unlimited material supply advantage.

Although cell lines are easy to work with, the physiological relevance of those studies might not be high. They do not show resemblance to human body metabolism and physiology, or even morphology. The immortalization and serial passaging cause several variations in genotype and phenotype of these cells. Due to the lack of morphological or functional features, cell lines might not be able to induce relevant biomarkers. Therefore, it is always better to validate cell lines before use to make sure they are not misidentified or contaminated.

For more info on advantages and disadvantages of primary cells in culture, read here.

Looking for primary cell culture for your experiments? Contact KOSHEEKA at info@kosheeka.com and procure tissue-specific human primary cells for your research.

How To Immortalize Primary Cells For Creating A Cell Line?

Primary cells can undergo a pre-determined and finite number of cell divisions in culture. With more passage in cell culture, primary cells enter a replicative senescence state, where morphology, gene expression, and metabolism alter. Therefore, scientists immortalize primary cells in vitro to study cell growth, differentiation, and senescence using continuous cell lines. Immortal cell lines made from primary cells are a powerful tool for biomedical investigators to research the biochemistry and cell biology of multicellular organisms for application in the research of cell biology, immunology, cancer biology, toxicology, and molecular biology.

Why Do We Need Cell Immortalization?

Primary cells reach senescence after limited generation and the process of frequently re-establishing fresh cultures from explanted tissues is tedious. Using immortalized primary cells or cell lines guarantees the consistency of the experiment’s materials. In addition to the capacity of extended proliferation, immortalize primary cells possess similar genotype and phenotype to their parental tissue. Moreover, many studies with hTERT-based immortalize primary cells have given differentiated cell types with tissue-specific features, differentiation-specific proteins. Along with these points, the ease of handling and maintenance of cell lines is a vital factor in cell biology research.

Cell Immortalization Strategies

Spontaneous Mutation

Some primary cells may be mutated and that can break the sequence of limited lifespan, leading to expanded cultivation and immortalization. This method of immortalization can be done in vitro and is called a spontaneous mutation. This method of creating immortalize primary cells is inefficient and the cells have the risk of transforming into tumor cells for most cases. So, tumor cells are supposedly the best examples of spontaneously immortalize primary cells, which may have undergone genetic changes to resist senescence.

kosheeka immortalized primary cells

Introducing Viral Gene to Override the Cell Cycle

Viral genes can affect the cell cycle by deregulating the biological brakes on the proliferative control of the cells. One way to use viral genes for immortalization is to use the simian virus 40 (SV40) T-antigen, while some procedures involve using viral oncogenes. To know more about how viruses can alter the cell cycle, read this research article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252338/

Viral Genes Cycle - Kosheeka

Telomerase Reverse Transcriptase (TERT) Expression

Telomerase is a ribonucleoprotein that can extend the DNA sequence of telomeres, which lets the cells undergo infinite cell divisions through evading the senescence process. This Telomerase Reverse Transcriptase (TERT) expression is generally inactive in most somatic cells, but when exogenously expressed, the cells are able to maintain sufficient telomere lengths for avoiding replicative senescence. This approach is the most common approach in current times.

Telomerase Reverse Transcriptase (TERT) Expression - Kosheeka

Combining Suppressor Cell Cycle and TERT Expression

In some types of cells, a single procedure of immortalization may not be helpful in getting immortalize primary cells. Thus, depending on different cell lines it would be better to combine methods of cell cycle suppressing and TERT expression to obtain immortalize primary cells.

Currently, TERT protein expression is being used in making immortalize primary cells as a combinational procedure along with viral gene and mutation procedure. Researchers involved in the work of cell immortalization should also take care of cell line quality-control considerations. For obtaining primary cells in order to research on immortalization and get cell lines, Kosheeka can help you with tissue-specific and species-specific primary cells upon inquiry at info@kosheeka.com

10 Amazing Facts About Cell Culture You Should Not Miss

Biomedical and clinical research would be inconceivable without progress in cell culture. This tool has become inevitably crucial in life sciences and has evolved much since the early 20th century, with its application in primary cell culture, 3D culture, tissue engineering, 3D bioprinting, and regenerative therapies. As a cell culture researcher, you must have enough expertise in your domain of research but cell culturing has a vast area and several facts remain scattered throughout the history.

In this article, we present such 10 lesser known facts that can help you exclaim and love cell culture more!

1# More than 32,000 papers rely on misidentified cell line data

In case of cell lines, misidentification is a relevant problem and studies estimate that over 32,000 papers report their data based on the work done on misidentified cell lines. The problem booms exponentially when a plethora of other papers cite these reports, leading to a contaminated pool of data (Horbach and Halffman, 2017).

2# 70% researchers fail to reproduce the data of experiments

A Nature survey suggested that 70% of more than 1,500 researchers were unable to reproduce clinical research experiments and 50% of them even failed to reproduce their own ones.

3# First successful cell culture: Frog nerve fibers

An American zoologist Ross Granville Harrison was able to grow animal cells outside the body, successfully in 1907. He grew frog nerve fibers using the hanging drop method in a lymph medium (Abercrombie, 1961).

Cell Culture

4# HeLa cell line was established without the consent of Henrietta Lacks

This might not be a grand surprise for many researchers that HeLa cell line was the first to be immortalized in 1951, but it is interesting that such a ground-breaking progress in the biomedical cell culture field was done without the consent of the human source: Henrietta Lacks or her relatives without her consent. This raised numerous concerns about ethical privacy and patients’ rights in research. 

5# The first synthetic mammalian cell culture medium was created in 1950

JF Morgan developed medium 199 in 1950 and it was the first synthetic media for mammalian cell culture (Morgan et al., 1950). This development helped in efficient medium for vaccine production and allowed large-scale manufacturing of polio vaccines in 1955. 

6# Cell cultures can be contaminated by plastics

Virus, bacteria, fungi, mycoplasma, and endotoxins are common contaminants in cell culture but little do we acknowledge the contamination by plasticizers from plastic instruments. These plasticizers can contaminate cell culture and alter cell physiology (Yao and Asayama, 2017).

7# Pluripotent stem cells can help in growing mini-brains

In 2013, researchers from the University of Vienna developed brain organoids or “mini-brains” in the lab using human pluripotent stem cells and a 3D matrix for support. The generated brain organoids showed different nerve cell types and structurally mimicked mammalian brains (Lancaster et al., 2013).

8# Fat tissues to heart: success of 3D bioprinting

In 2019, researchers from Tel Aviv University’s School of Molecular Cell Biology and Biotechnology developed a new protocol for generating 3D-printed thick and perfusable vascular hearts from a biopsy of fat tissue. The 3D-printed hearts anatomically mimicked the properties of the original heart but lacked functionality (Noor et al., 2019).

9# Supercooling can enhance organ ex vivo life

A 2019 article in Nature Biotechnology has shown that supercooling human livers at -4ºC for storage can prevent ice formation, thus extending the ex vivo life from 12 to 27 hours (de Vries et al., 2019).

10# The 3D cell culture market could reach nearly USD four billion in 2021

In 2016, the global 3d cell culture market was valued at 1 billion USD and according to BBC Research, it is going to reach 4 billion USD by 2021 with an AGR of around 30%.

There are several other interesting cell culture facts to know for a budding the researchers. For more such information, contact info@kosheeka.com. If your lab is looking for tissue-specific primary cell culture, you can also visit our website at http://kosheeka.com/

2D vs 3D Cell Cultures – What’s The Difference?

In the field of biomedical research, 2D cell cultures have extensively been used since the early 1900s but in recent times, the technology of 3D cell culture has boomed. The tussle of 2D vs 3D cell culture has received much interest from biomedical researchers and other science geeks. In this article, let us explore more regarding 2D vs 3D cell cultures.

What is 2D Cell Culture?

In 2D cell culture systems, cells are grown on flat dishes with coated surfaces to help them adhere and proliferate. These models are not representative of the in vivo cell environment as they do not mimic the microenvironment found in body tissues. Moreover, 2D cell culture testing is not always predictable for cell morphology and thus results in failure of drug discovery or research outcome rates. Also, as cells grow in 2D culture, they consume the media and exude metabolic waste, leading to damage of the cells by toxic waste and nutrition depletion.

2D Vs 3D Cell Cultures

What is 3D Cell Culture? 

Ross Granville Harrison adapted the hanging drop method to develop the first tissue culture and henceforth, this method has helped several studies in embryology, oncology, virology, genetics etc. The 1990s were a low time for 3D cell culture research but in the last decade, 3D cell culture market has seen an explosion with several capitalists and investors betting on 3D cell culture to be the key to clinical progress. This enormous faith is probably due to the varied application of 3D cell culture in cell therapy, drug discovery, toxicity, tissue engineering, organ bioprinting, and other biomedical domains. In 3D cell culture, the cells are grown in 3D structures by abolishing the monolayer system of growing cells by using hanging-drop-based, matrix-based, and agitation bioreactor-based methods. 

Advantages of 2D Cell Cultures

Even though the 2D vs 3D cell cultures debate still interests scientists, 2D cell cultures are still used for a majority of reasons: 

  1. 2D cell culture is less expensive than 3D cell culture models.
  2. 2D cell culture is well established and has been in use since the 1900s.
  3. More comparative literature data to explore cells.
  4. Easy cell culture observation and analysis.

Advantages of 3D Cell Culture

Even though 2D cell culture is easy to grow and maintain, 3D cell culture is the better choice in the dilemma of 2D vs 3D cell cultures. The difference between 2D cell culture and 3D cell culture can be best understood by the fact that 3D cell culture represents the human tissue outside the body while 2D cell culture only exists in a 2-dimensional monolayer, which is quite an inaccurate representation of the cell microenvironment and cell physiology.

The advantages of 3D cell culture include:

  1. 3D cell cultures are more physiologically relevant and predictive than 2D cultures. 
  2. 3D cell cultures are complex systems linked together by microfluidics and thus show better cell-ECM and cell-cell interactions.
  3. Better metabolic adaptation and flow integration make 3D cell culture more functionally relevant.
  4. Better simulation of conditions using microfluidics provides 3D cell culture advantage over 2D cell culture models.
  5. 3D cell culture reduces the use of animal models and thus are ethically relevant.
  6. 3D cell cultures are more realistic for developing tumor organoid models for research.

The transition from 2D cell culture to 3D cell culture in this debate of 2D vs 3D cell cultures is a popularly discussed scientific notion in recent times owing to the physiological research relevancy of the 3D models but a lot of research is still done with 2D cell culture practices due to the ease of culture maintenance. If you are working in cell culture domain and are looking forward to procure tissue-specific primary cells, contact us at info@kosheeka.com or visit our website at http://kosheeka.com/

Human Primary Cells Vs Immortal Cell Lines

In biomedical research and development, scientists mostly use cultured cell lines as models because they offer easy, inexpensive, and stable platforms. However, cell lines fail to grasp the microenvironment physiology that occurs in vivo. Human primary cells come to the rescue in this situation! Primary cell culture portrays the complex physiological cell behavior and therefore represents the in vivo microenvironment better. Once you decide on your hypothesis, it is up to your research requirement whether to use human primary cells or immortal cell lines to achieve the required research efficacy.

Human primary cells closely represent the human in vivo physiology and in the case of establishing experiment results, human primary cells offer a better and relevant cell culture model.

Types of Primary Cells

Human Primary Cells - Kosheeka

The figure above shows the different types of primary cells examples that can be isolated from a wide variety of body tissues. Human primary cells are isolated directly from body tissues and therefore retain the functional and morphological characteristics of their origin tissue. In comparison, several types of cell lines, that are formed by immortalizing human primary cells, display differences in morphological and genetic characteristics.

Human Primary Cells Vs Cell Lines

Human primary cells don’t live forever and undergo senescence. They have limited potential of self-renewal and differentiation. Late passages of human primary cells show morphological and functional changes and therefore behave differently under similar culture conditions. Different types of cell lines, on the other hand, can run for longer passages and do not undergo senescence. But identification of the authenticity of cell lines is a difficult task. In other aspects, cell lines are cost-effective and easy to culture or expand as compared to human primary cells. For some research experiments, this is supposed to be an added advantage.

Human primary cell cultures are easy to get contaminated and not easy to expand. Primary cell cultures often need experienced hands for good culturing practice. Other than these major points of difference, there are other small yet significant differences between human primary cells and cell lines, which as mentioned in the table below:

Human Primary Cells vs. Cell Lines

Last but not the least, cell lines are easy to procure but human primary cells lack easy availability owing to difficulty in their maintenance and culturing. But Kosheeka breaches this limitation with its wide variety of tissue-specific and species-specific primary cells for delivery. Visit their website at http://kosheeka.com/ or contact info@kosheeka.com for further inquiries.

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.

How Human Umbilical Cord-derived Mesenchymal Stem Cells Used In Research?

The human umbilical cord tissue was regarded as medical waste until 1991 when fibroblast-like cells were isolated from the human umbilical cord tissue. Further characterization of these cells revealed that they express adhesion molecules (CD44, CD105), integrin markers (CD29, CD51), and mesenchymal stem cell markers like SH2 or SH3. Pluripotency markers like Oct-4, SSEA-1, and SSEA-4 were also found on these cells besides the proof of differentiation into osteoblasts, chondroblasts, and adipocytes. These evidences determined the presence of human umbilical cord-derived mesenchymal stem cells. Since the first isolation of human umbilical cord-derived mesenchymal stem cells (UC-MSCs), more research was directed towards obtaining (MSCs) from the endothelial and subendothelial layer of the umbilical cord vein.

These cells were further shown to express mesenchymal stem cell markers CD29, CD13, CD44, CD49e, CD54, CD90, and HLA-I, besides having the ability to differentiate into cardiomyocytes. Some human (UC-MSCs) even show the presence of embryonic stems cell markers.

Human (UC-MSCs)

Research Application of Human Umbilical Cord-derived Mesenchymal Stem Cells (UC-MSCs)

Since their initial discovery and characterization, human (UC-MSCs) have been used in multiple cell therapies and clinical trials for targeting inflammatory disorders, cancer, neurodegenerative diseases, etc. There are over 400 registered clinical trials utilizing human (UC-MSCs). Recently, researchers have even used human (UC-MSCs) as a therapy for rheumatoid arthritis successfully to reduce related symptoms. Research studies further suggested that human (UC-MSCs) regulated patients’ autoimmune tolerance.

One of the most recent clinical trials of human (UC-MSCs) in tissue engineering application is based on the treatment of chronic skin ulcer treatment. In the study, human (UC-MSCs) in the form of bioscaffold mesenchymal stem cells are used to treat diabetic wounds owing to the anti-inflammatory, immunomodulatory and angiogenic properties. Researchers have also used human (UC-MSCs) to treat spinal cord injury in the form of bioscaffold mesenchymal stem cells to regulate the sustained release of cells at the injury site. These studies successfully showed the formation of new nerve fibers and the gain of electrophysiological activity.

Besides being a potential therapeutic tool in tissue damage disorders, human (UC-MSCs) have also been used in cancer research due to supposed anti-tumorigenic properties. In the case of Cancer Cell Lines and in-vitro models, human (UC-MSCs) are used as vehicles for targeted anti-cancer agent delivery to observe cancer cell growth inhibition.

Human umbilical cord-derived mesenchymal stem cells have become popular in clinical therapeutic research. Their accessibility and ease of cultivation make these cells an attractive tool for Stem Cell Therapy and tissue engineering besides bioscaffold-based modeling. If your lab is looking for tissue-specific stem cells, cancer cell lines, or primary cells culture, Kosheeka can help you get your hands on the best quality! Contact info@kosheeka.com with your inquiries.

Latest Development In PARP Inhibitors For Prostate Cancer Treatment

This article reviews the status of PARP inhibitors for prostate cancer treatment and focuses on recent developments in drug approvals.

How PARP Inhibitors help in Prostate Cancer Treatment?

Prostate cancer is a heterogeneous disease, often driven by specific genomic alterations. The defects in DNA repair/DNA-damage response pathways are critical to the risky malignant transformation of prostate cancer. The enzymes PARP, BRCA 1/2, and ATM play important roles in the malignant transformation of prostate cells and drug developers are taking advantage of this concept for devising prostate cancer cure. In this concept, where one gene is inactivated by mutation, the other is inactivated by a drug. This approach of PARP inhibitors is successfully used in tumors having DNA damage defects and therefore can be used for prostate treatment.

PARP Inhibitors

Two PARP Inhibitors for the Cure of Prostate Cancer

Recently, the FDA approved two PARP inhibitors for prostate cancer treatment—Rucaparib and Olaparib. These drugs were found to delay cancer progression in men with metastatic prostate cancer.

Rucaparib was the first of the two approved PARP inhibitors and is indicated for the cure of adult patients with metastatic prostate cancer having a BRCA mutation. The approval for rucaparib for prostate cancer treatment was supported by a multicenter, single-arm trial known as “TRITON2”. Shortly after the Rucaparib approval, the FDA announced the approval of Olaparib based on data from the PROfound clinical trial. 

Ongoing PARP Inhibitor Clinical Trials for Prostate Cancer

Some of the ongoing PARP inhibitor clinical trials include:

PROpel [Olaparib]: This is a Phase III trial for testing Olaparib as a 1st-line medicine in genetically unselected metastatic prostate cancer treatment in combination with Abiraterone.

TALAPRO- 1 [Talazoparib]: This is an open-label, Phase II trial evaluating Talazoparib in metastatic prostate treatment of cancer with DNA damage defects after 1–2 chemotherapy regimens.

TRITON3 [Rucaparib]: This is an open-label Phase III trial comparing Rucaparib with a physician’s choice of therapy for metastatic prostate cancer treatment associated with an HRR gene defect. 

MAGNITUDE [Niraparib]: This is a Phase III trial evaluating Niraparib in combination with Abiraterone and Prednisone in adults for metastatic prostate cancer treatment.

More Information on Using PARP inhibitors for Cancer

Most of human prostate cancer pathogenesis tend to form spheres and adaptation of cancer cells or cancer stem cells to non-adherent culture conditions may be a crucial factor in this regard. In prostate cancer pathogenesis, the holoclone-forming cells, which are adherent and highly clonogenic, have been associated with stem cell phenotypes, suggesting their inclination towards being tumor-initiating cells with stem cell-like features of strong self-renewal and pro-angiogenic capability. Accordingly, in these prostate cancer sphere models, the expression of putative cancer stem cell markers such as ALDH1A1, CD44, CD133, showed a strong correlation with progression and metastasis.

Researchers are working towards using PARP inhibitors to inhibit cancer metastasis in these prostate cancer sphere models and moreover, reconstruction of tumorspheres using bioscaffold mesenchymal stem cells are also being sought to understand the mechanism of cancer resistance. In addition to being targeted for prostate cancer treatment, PARP1 is also reported to repress NKG2DLs expression and mediate immune evasion of leukemic stem cells (LSCs) in the case of acute myeloid leukemia. Therefore, PARP1 inhibition could restore NKG2DL expression and promote their immune-mediated clearance. Thus, the multifactorial functions of PARP1 in immunomodulation and immune escape of cancer adult stem cells may pave the way for novel therapeutic strategies in the clinic.

PARP inhibitors are exciting and novel precision therapeutics for prostate cancer treatment and more research is underway to establish further efficiency of PARP inhibitors. If your lab is working on prostate cancer treatment research and you are looking for cancer cell lines or disease-specific primary cell culture, contact us at info@kosheeka.com with your inquiries.

Major Discoveries In Animal Cell Culture Media

History of Animal Cell Culture Media

In 1882, Sydney Ringer developed Ringer’s solution, a balanced salt solution with composition close to that of bodily fluids, to keep beating frog hearts after dissection. This is considered as the first witnessing of in vitro animal tissue cultivation. In the wake of Ringer’s report of the animal Cell Culture Media solution, balanced salt solutions were developed including Locke’s solution, Tyrode’s solution, Krebs–Ringer bicarbonate solution, Earle’s solution, and Hanks’ solution. The composition of these balanced salt solutions generally included only inorganic salts, sometimes with glucose. The pH, osmolarity, and salt concentrations were calibrated and optimized to physiological solutions to keep cells and tissues in vitro successfully for a few days.

Gradually, the focus was shifted from making animal cell culture media to maintaining the cells for more time as the cells did not usually survive for more days and rarely showed healthy proliferation. In 1907, Ross G. Harrison did a successful monitoring of an apparent nerve fiber outgrowth for several weeks in freshly drawn lymph fluid from an adult frog. In the history of Animal Cell Culture media, this was considered as a milestone.

The first success of animal cell growth culture by Harrison inspired Montrose T. Burrows to work under him and during his work, he found that plasma is better suitable for animal cell culture from warm‐blooded animals instead of lymphatic fluid. Burrows successfully cultivated chicken embryonic cells by using plasma and later went ahead with mammalian cell culture. In 1913, Carell discovered that adding embryonic extract to plasma can increase proliferation and culture period of fibroblasts. In due course, scientific inquiry regarding the components of plasma, lymph, and embryonic extract was popular to find the factors affecting survival and growth of animal tissues and cells. 

Warren H. Lewis and Margaret H. Lewis, demonstrated that the Locke–Lewis solution (modified Locke’s solution with additional amino acids, glucose, and nutrients) is more effective for embryo cell culture than simple balanced salt solutions. They reported that glucose and partially hydrolyzed proteins effectively promote the animal cell culture growth. In 1940, Wilton R. Earle et al. used carcinogens to successfully surpass the Hayflick’s limit to create immortal mouse Fibroblasts and due to the emergence of established continuous cell lines, scientists started examining and quantifying the differences in the animal cell culture media effects. The need for advanced animal cell culture media thus crept up and scientists started looking intently into understanding and determining the specific components in animal cell culture media instead of naturally derived media components of unknown composition.

Animal Cell Culture - Advancells

The first strategy to understand was using dialyzed serum and adding defined components for the animal cell culture support while the second strategy involved creating the formulation using exclusive definite media components. The first strategy was popularly used by Fischer and he found that low‐molecular‐weight fraction (amino acids being the key component) was essential for the enhanced survival of animal cells. On the basis of Fischer’s method, Harry Eagle studied the minimum necessary amounts of low‐molecular‐weight components used for animal cell growth culture, in 1995. Upon finding that glucose, 13 amino acids, and 8 vitamins are necessary, he developed the minimum essential medium (MEM). This composition was then modified by several scientists like Dulbecco, Vogt, Stanners, Iscove, and Melchers. Later, Thomas A. McCoy et al. suggested the use of pyruvate for specific cells in his 5A medium but the Roswell Park Memorial Institute (RPMI) modified it further in terms of its Ca and Mg concentrations to come up with RPMI 1640 used in lymphocyte culture.

The other strategy was popularly researched by Philip R. White, who developed a chemically defined medium composed of glucose, inorganic salts, amino acids, iron, vitamins, and glutathione, without protein. Some researchers suggested that this media still needed 10%‐20% serum for obtaining similar animal cell culture reports to White. Later, Connaught Medical Research Laboratories (CMRL), developed Medium 199 and further went ahead to compose a chemically defined medium, CMRL1066, consisting of 58 components after several modifications. Following that, other chemically defined mediums like NCTC109 and MB 752/1 were prepared to optimize animal cell culture. 

In 1963, G. Ham developed Ham’s F‐10 medium with two serum protein fractions (albumin and fetuin) instead of serum, and successfully cultured Chinese hamster ovary (CHO) cell lines. However, its proliferative capacity was less than that of CHO cell lines in serum‐containing media. To further elevate his research, Ham replaced albumin and fetuin with low‐molecular‐weight linoleic acid and putrescine to develop Ham’s F‐12, a synthetic defined animal cell culture medium. 

With time, growth factors like nerve growth factor, epidermal growth factor, insulin‐like growth factor, fibroblast growth factor (FGF), platelet‐derived growth factor, and transforming growth factor (TGF) were discovered to increase cellular proliferation but their effect on animal cell culture proliferation was always enhanced with some amount of serum in the culture media. But researchers were bent on discovering optimum serum-free medium and that led to some interesting discoveries. Ham discovered that selenite (trace element) is required for the serum‐free culture of human diploid cells whereas Guilbert and Iscove showed that transferrin and albumin combination, besides selenite, can be a good serum substitute. Prompted by these animal cell culture media discoveries, several attempts were made to optimize cell culture media as per researcher’s interest and application

Significant Fuel for Animal Cell Culture Research

  • In 1982, clinical application of recombinant human insulin expressed in E. coli led researchers to find different expression systems as glycosylated proteins were not possible in prokaryote systems. Therefore, researchers started realizing the application of animal cell culture in genetic engineering and the production of recombinant proteins. Among several host cell lines used, CHO and NS0 cells gained popularity in the field of biopharmaceutical manufacturing due to a few reasons: (a) technological advances in scaled-up culture methods (b) knowledge regarding virology of the cell lines and (c) advances in high‐expression derived sublines. The production efficiency in these cases required that the animal cell culture medium contains none or a minimal amount of natural biological ingredients, like serum, as they hamper protein purification. This is one research direction that led scientists to come up with several culture media modification strategies based on medium component and byproduct concentration, genomics and proteomics-based approaches, and host-cell modifications. Due to the patent rights and commercial value of these media compositions, biopharma companies have not disclosed them over years. 
  • The biomedical community is well-aware of the special place that embryonic stem cells and pluripotent stem cells hold due to their usefulness in basic and clinical regenerative medicine studies. Owing to the huge demand, the need for culturing these cells in a low‐cost and highly productive way led to modifications in animal cell culture media. The animal cell culture media involved a layer of feeder cells in a serum‐ or KSR‐supplemented medium but soon, research began in order to come up with feeder-free media like E8 medium. Further research is being conducted to develop more media compositions for the long‐term growth of stem cells using low-molecular weight compounds. 
  • In 1978, the first successful in vitro fertilization application used Ham’s F-10 media with serum supplements for human zygote culture. Later scientists found that hypoxanthine and the trace elements in Ham’s F‐10 negatively affected the embryos as metabolism of a pre‐implantation embryo differs from that of somatic cells. With further research, the usually preferred composition of the embryo culture media was similar to G1/G2 medium or the KSOMAA medium for optimum cell culture practice.

Aside from the mentioned milestones, there were numerous other applications like viral culture technique, cancer research, and acellular matrix bioscaffold model, which led to major turning points in the course of developing optimum animal cell culture media to keep up with the ever-evolving pace of scientific and clinical studies.