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Summer Research Fellowship Programme of India's Science Academies

Training on basic cell culture technology

Alka Thakur

Center for Basic Sciences, Pt. Ravishankar Shukla University, Amanaka, Raipur 492010

Dr. Mohan R. Wani

Scientist-G, National Centre for Cell Science, Savitribai Phule Pune University Campus, Pune 411007

 Abstract

Cell, the fundamental unit of life, is an incredibly useful tool in biological research. Cell culture is basically survival and proliferation of cells in an artificial environment (in vitro). The areas in which the cell culture is used are production of monoclonal antibodies, viral and insect vaccines, enzymes, hormones, and growth factors. Today's progress in cell culture research has resulted in the creation of concept of regenerative medicine. Studying cell culture requires unique operating circumstances. A cell culture laboratory must have sterile environment to avoid contaminations. I learned isolation and culture of mouse bone marrow-derived mesenchymal stem cells. I have taken primary cells, also known as finite cells, that were directly isolated from mouse, fed these cells every third day with complete alpha-minimal essential media (α-MEM +10% FBS) and observed them under the microscope. If cultured under the optimum conditions, primary cells will grow and proliferate, but they are able to do so up to a finite number of times. After growing them in culture for seven days, cells got 80% confluent. If cells are not sub-cultured at this stage, they get deprived of nutrients and space which can lead to cell death. Sub culturing/passaging means transferring some or all of the cells from a previous culture to fresh growth medium. When my cells got 80% confluent, I sub-cultured them using TPVG (Trypsin phosphate versene glucose), thus providing more nutrient and space for them to grow and proliferate under optimum conditions. I have passaged these BM-MSCs thrice and still maintaining them in culture. In future I will learn RNA isolation from these cell and synthesize cDNA.

Keywords: cell culture, passaging, sub culture, aseptic techniques

Abbreviations

Abbreviations
FBS Fetal bovine serum
α-MEM Alpha-minimal essential media
RNA Ribonucleic acid
MSCs Mesenchymal stem cells
DNA Deoxyribonucleic acid
P3 Passage 3
Min Minute
Rpm Rotation per minute
Ml milli liter
Hrs Hours
TPVG Trypsin phosphate versene glucose
DEPC Diethyl pyrocarbonate
µl micro liter
dNTPs Deoxyribonucleotide triphosphate
NF H2O Nuclease free water
DTT Dithiothreitol
RT Reverse transcriptase

INTRODUCTION

Cell Culture

Cell, the fundamental unit of life, is an incredibly useful tool in biological research.Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental i.e artificial condition (in vitro) (Hudu, 2016). They serve as an essential scheme for understanding physiological procedures, screening and used in medical treatments of poisonous or therapeutic compounds. In addition, cells, along with a variety of other uses, play a significant role in functional enzyme, growth factor, and vaccine production. Ross Harrison (1907) developed the first technique of cell culture in vitro in the first decade of the twentieth century, and Burrows and Carrel (1910) improved the cell cultures of Harrison. The basic principles for in vitro plant and animal cell cultures were created in the mid-twentieth century and human diploid cell lines, were established on the basis of knowledge about the cell cycle and gene expression regulation. Cell culture corresponds to removing cells from an animal or plant in a favorable artificial surrounding and their subsequent development. The cells can be separated directly from the tissue and disaggregated before cultivation by enzymatic or mechanical means, or they can be obtained from an already developed cell line or cell strain. After the cells are separated, primary culture is form, the primary culture is the phase at which culture occupies all the available substrate or spaceand proliferates under the suitable condition. After the confluent stage the cells must be sub-cultured (i.e. passaged) by transferring them to a new vessel with a fresh growth medium in order to provide more space for continuous growth. The normal cells generally divide only a few times before they lose their capacity to proliferate, known as finite cell line; which is a genetically determined event called as senescence. However, some cell lines become immortal or acquire the ability to divide indefinitely, known as continuous cell line. A cell culture developed from a single cell and therefore consists of cells with a uniform genetic make-up is known as cell line. A cell line is a permanently established cell culture that will proliferate indefinitely when provided with appropriate fresh medium and space. Cell lines are used to express different types of proteins in mammalian cells. Most cells are anchoring dependent and must be cultivated in a solid or semi-solid substrate (adherent or monolayer culture), while others float in a culture medium (suspension culture). The condition for cell culture varies widely for different types of cells, but the artificial environment in which cells are usually grown occurs in a suitable vessel consisting of a substrate or medium that provides basic nutrients like (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones and gasses (O2, CO2).

Aseptic Conditions

Aseptic technique means to exclude contamination. Successful cell culture depends on the maintenance of cells, free from contamination by microorganisms such as bacteria, fungi and viruses. Non-sterile supplies like media and reagents, loaded with airborne microorganisms, unclean incubators, and dusty work surfaces are all sources of biological contamination. Aseptic technique, aimed at creating a barrier between environmental microorganisms and sterile cell culture, and is based on a set of methods for reducing the risk of contamination from these sources. Aspects of aseptic technique are a sterile area of work, good personal hygiene, sterile reagents and media.

Media

One of the important steps in cell culture technique is the selection of the culture media, which provides the physical and chemical conditions similar to those in the natural environment for the development of cells that are essential for in vitro adhesion, reproduction and cell survival. Although initial studies on cell culture were conducted using natural media extracted from tissue extracts and body fluids, the need for standardization, media quality, and enhanced demand resulted to the development of defined media. The three basic classes of media are basal media, reduced-serum media and serum-free media, which vary in their requirement for serum supplementation. Serum is a complex mix of growth factors,albumins and growth inhibitors (Lane & Miller, 1976). Serum is one of the most significant parts of cell culture media and acts as a source of proteins, amino acids, vitamins (especially fat-soluble vitamins such as A, D, E, and K), carbohydrates, growth factors, minerals,lipids, hormones and trace elements. Serum from bovine sources of fetal and calf is frequently used to promote cell growth in culture. Fetal Bovine (FBS) serum is most widely used serum for cell line and cloning. I used complete alpha-minimal essential media [α-MEM (Sigma-Aldrich) +10% FBS (Gibco)] as media.

Cell Counting

The hemocytometer is a counting-chamber device invented by the 19th century French anatomist Louis-Charles Malassez and usually used for counting blood cells. A hemocytometer is a thick glass microscope slide with a grid of middle engraved perpendicular lines. The grid has specified dimensions so that the area covered by the lines is known, enabling the number of cells to be counted in a specific solution volume. To differentiate between dead and viable cells, the sample is diluted with a specific stain, such as Trypan blue. This technique of staining, also known as dye exclusion staining,this dye selectively penetrates dead cell membranes, coloring them blue, while it is not absorbed by live cell membranes, thus excluding live cells from staining. Dead cells would appear as dark blue when viewed under a microscope. The most common type of hemocytometer is engraved with "H" shape in the middle, enclosing two separate mirror-like polished grid surfaces and providing the mounting area for the cover slip. The Neubauer counting chamber is a well used form of hemocytometer.

RNA Extraction

 RNA (Ribonucleic Acid) extraction is the purification of RNA from biological samples. The ubiquitous presence of ribonuclease enzymes in cells and tissues, can readily degrade RNA, and complicates the process. Several methods are used in molecular biology to isolate RNA from samples, here I use TRIzol reagent to isolate RNA from mesenchymal stem cells (MSCs). TRIzol is a chemical solution that is used to extract DNA (Deoxyribonucleic Acid), RNA and cell proteins. TRIzol is light susceptible and often stored in a dark-colored, foil-covered glass container. TRIzol solubilization and extraction is recently developed, general method for deproteinizing RNA. This method is particularly advantageous in situations where cells or tissues are enriched in endogenous RNases or when separation of cytoplasmic RNA from nuclear RNA is impractical. The addition of chloroform after solubilization leads to separation of stage, where the protein is extracted into the organic stage, the DNA resolves at the interface, and the RNA is maintained in the aqueous stage. It is therefore possible to purify RNA, DNA, and protein from a single sample (hence the name TRIzol).

cDNA (Complementary DNA)

cDNA means complementary DNA or copy DNA. RNAs being unstable, brittle and very susceptible to degradation, by the ubiquitous RNases. To combat this, mRNA encoded biological information is stored in a more stable form of nucleic acid, i.e. DNA.Consequently, the cDNA is prepared from RNA that stores the entire sequence of mRNA. Working with cDNA is more convenient as compared to mRNA. This cDNA can be used for several subsequent researches on genetic and molecular biology. cDNA can be single stranded or double stranded. In genetics, complementary DNA (cDNA) is synthesized from a single-strand RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the reverse transcriptase (RT)enzyme. Reverse transcriptase (RT) is a RNA-dependent DNA polymerase. It operates on a single strand of mRNA. The reverse transcriptase uses mRNA as a template to generate its complementary DNA based on the pairing of base pairs of RNA. The sequence of cDNA is complementary to the sequence of RNA. This enzyme perform reactions in the same way as DNA polymerase. Unlike RNA, it is easy to clone DNA molecules these are called 'cDNA clones’.

OBJECTIVE

Cell culture is an important technique in both cellular and molecular biology as it provides the best platform for studying normal cell physiology and biochemistry. In order to understand an organism or given tissues, it is important to understand how its cells work, through cell culture. Whatever is learned about in vitro cells is representative of what is happening to the organism /tissue.

The main objective of this project was to learn basic cell culture techniques such as Isolation of mouse BM-MSCs, cell harvesting, cell culture maintenance, subculture / cell passage, cell counting, and RNA isolation, which is very important before initiating any specific cell study.

LITERATURE REVIEW

With the advancement of biochemistry, molecular biology, cell biology and other areas of biological knowledge has been possible to introduce new techniques to produce different cell lines, characterize and differentiate them in order to perform studies of cell interactions, host-parasite interaction, and so on. The cell culture has been indispensable to study virology and has made obtaining virus by using special animals since they provided large numbers of cells suitable for virus isolation, facilitated control of contamination with antibiotics, clean-air equipment, and helped decrease the use of experimental animals and virus isolation in cell cultures (Lindenbach et al. 2005; Leland et al. 2007). Cell cultures in cancer drug discovery have been very useful and new techniques are developed every day. Furthermore, plant cell cultures (Mokili et al. 2012), vaccine production, and biotechnology studies from drug production in bioreactors (interferon, insulin, growth hormone, etc.) have also been made (Li et al. 2010). Otherwise, cell culture applications in pharmacology and toxicology studies are testing the effect of different drugs, interactions of drug-receptor type, resistance phenomena, cytotoxicity, mutagenesis, carcinogenesis, among others. One example is the use of three-dimensional (3D) cell culture models (Smalley et al. 2008) which could be non-adherent (anchorage-independent) or adherent to a substrate (anchorage-dependent). In the 3D anchorage-independent culture the aggregation of cells can be achieved by using low-attachment plates and through coating surfaces (e.g., poly-hydroxyethyl methacrylate and agarose) (Friedrich et al. 2007).The primary goals for developing 3D cell culture systems vary widely from engineering tissues for clinical delivery through to the development of models for drug screening (Haycock, 2011). Another example of application rather studied in recent years has been the tissue engineering, for example, the production of tissue in vitro as skin or cartilage for treatment of burns, autografting, differentiation and induced differentiation (Naderi et al. 2011).

METHODOLOGY

I began my work with isolation of bone marrow cells from the 6–8 weeks old Balb/c mice and use these cells to learn the basics of cell culture technique like subculture/passage, cell counting on haemocytometer. I maintained bone marrow-derived MSCs, upto P3 (passage 3) and used these cell for the further experiments, such as RNA isolation & cDNA synthesis. To ensure expected cell health and growth, they are daily microscopically checked. The procedures that I performed for above techniques are as follows:-

Isolation and Maintenance of Mouse BM-MSCs

Bone marrow harvesting

To isolate cells, six to eight weeks old Balb/c mice were chosen to harvest bone marrow cells. By cervical dislocation mice were sacrificed and aseptically long bones is removed such as femur, tibia and humerus and kept in minimal essential media (αMEM) followed by removal of attached tissues from bone with sterile forceps and scissors,then bone epiphysis was cutwith sharp sterile scissors/scalpel and marrow tissue was flushed out into sterile petriplate by slowly injecting media from 23-gauge needle through the bone cavity,flush the bone marrow until the flow through bones turns white, all these procedure is performed under the hood to avoid contaminations. The suspension of the bone marrow cell was centrifuged for 7 min at 1500 rpm and cell pellet is obtained. With 10% FBS and penicillin and streptomycin (100U/ml), cell pellets were resuspended in 1ml αMEM. Cells were placed in a 25cm2 (T25) cell culture flask with 5ml of medium and incubated with 5% CO2 incubator at 37°C.

Cell passage/subculture

 For proper growth of cell, media should be changed after 72 hrs by discarding supernatant with non-adherent cells followed by addition of new media. On the 7th day, cells reach to 70–80 % confluent stage clearly representing the spindle-shaped morphology of adherent MSCs. After the cells attained 70-80% confluence stage in culture flask, to subculture MSCs, trypsinization was performed. The first step in sub-culturing adherent cells is to detach them from the surface of the culture vessel by enzymatic (TPVG) or mechanical means (scraping). Here TPVG is used to perform passaging. To transfer the cells into another flask, supernatant was discarded from the flask, cells were washed with2 ml TPVG followed by gentle rolling to ensure trypsin contact with all cells and 2 min of incubation. To remove maximum cells gently tap the flask. As soon as cells have detached add some culture media (containing serum) to the flask this will inactivate the trypsin.

Cell counting

For counting cells I used hemocytometer. Hemocytometer is divided into 9 major squares of 1mm x 1mm size. The four corner squares (identified by the red square in figure 1 below) are further subdivided into 4 x 4 grids and one square contain 16 smaller squares. The height of the chamber formed with the cover glass is 0.1 mm, so a 1 mm x 1 mm x 0.1 mm chamber has a volume of 0.1 mm3 or 10-4 ml.

hemocytometer.jpg
    Hemocytometer diagram indicating one of the sets of 16 squares that should be used for counting.

    For cell counting the cells were pellet down by centrifugation, resuspended in 1 ml of complete medium and 10µl of the suspension is added in 90µl trypan blue. To avoid contamination glass hemocytometer and cover slip,should be cleaned with 70% ethanol. 10µl of trypan blue-cell suspension is loaded on one of the chambers of the hemocytometer by closely touching the cover slip at its edge with the tip of the micro pipette and letting each chamber to fill with capillary action and observed under the microscope and count all the cells in the four 1 mm corner squares.

    RNA Extraction

    To isolate RNA, firstly cell pellets were obtained from culture containing atleast 105 cells, 500μl TRIzol reagent was added to disrupt and break down the cells and cell compartments, 100μl chloroform was added to separate the solution into different phases and vigorously mixed for 15 sec and left at RT (room temperature) for 2–3 min, centrifuged at 12000g for 15min at 4°C.At this point, there will be three layers in tube:

    (I) Top layer is clear and aqueous contains RNA,

    (II)Middle layer/ interphase is white and contains precipitated DNA,

    (III)Bottom layer is pink organic phase contain protein and lipids.

    TRIzol phase.jpg
      : Phase Separation using TRIzol

      Transfer the aqueous phase to the new eppendorf tube, add 250μl of isopropanol to precipitate the RNA, white turbid ring were observed; leave the aqueous phase of RNA in ice for 1hr, centrifuge at 12000g for 10min at 4°C.Decant the supernatant and wash the RNA pellet with 75% ethanol (prepared in DEPC (Diethyl pyrocarbonate) treated H2O); centrifuge it at 7500 g for 5min at 4°C.Decant it and air dry pellet to remove traces of ethanol and then resuspend the pellet in 10μl DEPC treated H2O followed by incubation at 65°C for 5min, and then quantify it using NanoDrop (ND-1000) spectrophotometer and stored at −20°C for further use.

      cDNA Synthesis

      Above extracted RNA was used to synthesize cDNA, which requires two mix:-

      Master mix and Water mix and below is a protocol for the following:

       

      Master Mix:

      Measurements  for Master mix
      Component Volume
      dNTPs (10mM) 2µl
      Primer(Random Hexamer) 1 µl
      RNA 2 µl
      NF H2O 7 µl
      Total Volume 12 µl

      Incubate the master mix in heat block for 5min at 65°C for denaturation of RNA secondary structure, then keep it on ice for 5–10min for annealing of primer; add water mix in the above mix and incubate for 1hr at 42°C for extension of strand followed by incubation at 80°C for 3min to inactivate the RT enzyme. Sum of volume of master and water mix should be 20µl.

      Water Mix:

      Measurments for water mix 
      Component Volume
      Buffer(5X) 4 µl
      DTT(Dithiothreitol) 1 µl
      NF H2O 2.5 µl
      RT Enzyme 0.5 µl
      Total Volume 8 µl

       RESULTS AND DISCUSSION

      Bone Marrow Harvesting

      After isolation, bone marrow cells seeded into culture flask (T25) shows spherical morphology. It was noted that cells started to adhere to a culture vessel after 72 hours. Media replacement separated majority of non-adherent cells from the culture flask and allowed more homogeneous MSCs to grow. On the 7th day, 70%–80% of the surface of the culture vessel was covered with spindle shaped MSCs expressing that confluence stage is reached.

      Cell passage/subculture

      Cellswere passaged through trypsinizing. Under the culture condition, cells usually remained highly morphologically heterogeneous before P2, with convex round, convex spindled and flattened spindled shapes. The proportion of flattened spindle-shaped cells was gradually elevated with the increase of cell passage number.The reason for this is that passaging tends to remove non-adherent cells leads to an increasing number of more adherent MSCs and gradually, a pure culture.

      microscope images.jpg
        Images of mouse BM-MSCs at different stages (A)Cells seeded in culture flask after harvesting at 10x, (B)Passage 1 MSCs at 10x, (C)Passage 2 MSCs at 10x ,(D)Passage 3 MSCs with 70-80% confluence at 10x.

        Cell Counting

        Systematically cells are counted in selected squares and cell concentration can be calculated from the following formula:

        Total no. of live cells=Avg. no. of cell count X Dilution factor X104

        Here dilution factor is 10 and 104 is the volume. In Passage (P1, P2, P3) 6.75 * 105cells were counted and transferred into one 25cm2 (T25)cell culture flasks with αMEM and incubated.

        RNA Extraction

        To quantify RNA (Ribonucleic Acid) NanoDrop (ND-1000) spectrophotometer was used. The absorbance spectrum of the RNA sample below indicates pure RNA, close to ideal A260/280 ratio but due to contamination A260/230 ratio was lower than expected and the concentration was within the reliable range of the Nanodrop ND‐1000 spectrophotometer.

        result RNA.jpg
          Quantification of RNA using NanoDrop

          260/280 Ratio-The ratio of absorbance at 260 nm and 280 nm was used to assess the purity of RNA. A ratio of approximately 2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower, then it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.

          260/230 Ratio-This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” RNA are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230nm, carbohydrates and phenol all have absorbance near 230 nm.

          cDNA (ComplementaryDNA)

          cDNA is synthesized from the RNA and to quantify it one can use NanoDrop. cDNA (Complementary DNA) can be used as gene probes or in gene cloning or in the creation of a cDNA library.

          CONCLUSION AND RECOMMENDATIONS

          Cell culture systems are indispensable tools for basic research and a broad variety of in vitro clinical studies. We can obtain products from these cultured cells or we can transplant them to treat some diseases. Researchers require high effectiveness and higher throughput cell-based testing; therefore, the cell culture used, must fulfill demanding requirements for performance, quality, accuracy and security. But aseptic techniques are still key point. A simple biological contaminant can easily ruin all these perfect studies. Cell culture holds great potential for groundbreaking applications in cell therapy and regenerative medicine and to advance our understanding of human biology. Successful cell cultures require optimized protocols, high-quality reagents and proper handling.

          REFERENCES

          Hudu, S. (2016). Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases. Journal of Clinical and Diagnostic Research

          Lane, B & Miller, S (1976) Preparation of large numbers of uniform tracheal organ cultures for long term studies. I. Effects of serum on establishment in culture. In Vitro; 12:147-54.

          Lindenbach, B.D., Evans, M.J., Syder, A.J., Wölk, B., Tellinghuisen, T.L., LIU, C.C., Rice, C.M.et al (2005). Complete Replication of Hepatitis C Virusin Cell Culture. Science, 309(5734): 623-626.

          Leland, D.S., Ginocchio, C.C. (2007). Role of cell culture for virus detection in the age of technology. Clin. Microbiol. Rev., 20(1): 49-78.

          Mokili, J.L., Rohwerf, Dutilh B.E. (2012). Metagenomics and future perspectives in virus discovery. Curr. Opin. Virol., 2(1): 63-77.

          Li F., Vijayasankaran, N., Shen, A., Kiss, R., Amanullah, A. (2010). Cell culture processes for monoclonal antibody production. MAbs, 2(5): 466-477.

          Smalley, K.S.M., Lioni, M.,Noma, K., HAASS, N., HERLYN, M. (2008). In vitro three-dimensional tumor microenvironment models for anticancer drug discovery. Expert Opin. Drug Discov., 3: 1-10.

          Friedrich, J., Ebner, R., Kunz-Schughart, L.A. (2007). Experimental anti-tumor therapy in 3-D: Spheroids-Old hat or new challenge?.Int. J. Radiat. Biol., 83: 849-871.

          Haycock, J.W. (2011). 3D cell culture: a review of current approaches and techniques. Methods Mol. Biol., 695:1-15.

          Naderi, H., Matin, M.M., Bahrami, AR. (2011). Critical issues in tissue engineering: biomaterials, cell sources, angiogenesis, and drug delivery systems. J. Biomater.Appl., 26(4):383-417.

          ACKNOWLEDGEMENTS

          I would like to extend my heartiest gratitude to respected Dr. Mohan R. Wani, Scientist-G, National Centre for Cell Science, Pune for his wonderful guidance, encouragement and constant support at every step of the project work.

          I am grateful to all lab members with whom I had the pleasure to work during this project. Each of the members has provided me extensive personal and professional guidance and taught me a great deal about scientific research and life in general.

          I would like to thank my parents & brother whose love and guidance are with me in whatever I pursue.

           

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