Rohit Sagar Assistant Professor Deshbandhu college What is cell culture ?
• Cell culture is a technique which involves isolation of cells from animal/plant body i.e. from their natural environment (in vivo) and practicing to grow isolated cells in cell specific media in plastic flask or petri dish in a controlled environmental artificial condition (in vitro).
• Cell culture means to keep cells alive and grow in an in vitro condition in a nutritive media which are widely used for research and diagnosis of different pathogens and to understand the function and mechanism of operation of many cells. Types of cell culture • Primary culture Primary culture is the cell culture system that is formed by culture cells directly obtained from tissue and proliferate them until they occupy all of the available substrate (i.e. reach Confluence). For primary culture, the first step is to isolate the tissue from organ and splitting of cells from tissues. This can be achieved by trypsin treatment. • Then grow cells in freshly prepared cell specific medium and incubate the flask containing cells in incubator providing suitable environmental condition for the growth of cells. • After the first subculture, the primary culture is known as a cell line or subclone. •Secondary culture • When cells from primary culture on confluency are isolated and cultured in new media, it is called as secondary culture. • Secondary culture is also known as subculture or splitting of cells. • Subculture allows fresh nutrients and more space for the expansion of the cells. • Cells from primary culture are splits by trypsin/EDTA treatment. • Trypsin which is a serine protease digest the extracellular protein or matrix protein so that cells get free. • EDTA which is a chelating agent chelates calcium ion because calcium helps in cell adhesion. Finite Vs Continuous Cell Line
• Normally, cells divide to a limited times, after that loose their proliferation ability (senescence) these cell line are known as Finite cell line. • Cell line which are chemically or virally transformed (transformation) acquires the ability to proliferate indefinitely, these cell line is called continuous cell line. • Contact inhibition and anchorage dependence and low growth rate are the characteristics of finite cell line. • Absence of contact inhibition and anchorage dependence and high growth rate are some attributes for continuous cell line. • Finite cell line grow as monolayer culture i.e cells cover the bottom of the culture vessel uniformally. • Continuous cell line may be grow as monolayer or suspension culture (cells do not adhere but remain suspended in culture media). Oyeleye et al, 2016 Cell lines generally used in lab SF9 cell line (Spodoptera frugiperda) BHK-21 cell line at different confluency
Vero cell line . ATCC means American Type Cell Culture Requirement of cell culture • A substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals). For enhancing growth and • Growth factors (Fetal Bovine Serum) proliferation of cells • Hormones
• Gases (O2, CO2) • A regulated physico-chemical environment (pH, osmotic pressure, temperature). • Bio safety cabinet/Laminar hood/ BOD • Serological sterile pipette, • Trypsin/EDTA solution • Phosphate buffer saline • T-25/T-75 culture flask. • Penicillin/streptomycin solution (required to prevent bacterial contamination). Animal cell culture media
(Yao et al, 2016) DMEM MEDIA COMPOSITION
Note- 10% DMEM media is required for normal cell culture like Vero or C6/36 or BHK-21 cell lines. ✓ So to make 10%, we have to add FBS and streptomycin/penicillin solution, now we have complete DMEM MEDIA which is ready to use for culturing ✓ All other components are already added in media so no need to add others. Major equipments required for cell culture
Bio Safety cabinet
Cell culture incubator Sub-culturing of cells on confluency Broad category of animal cell culture media Overview of how cell culture is performed in culture lab Procedure for maintenance of animal cell culture like Vero cell line Things to be done before starting cells passaging 1) First of all we have to give UV radiation to culture hood for sterilisation. 2) Media which was kept in 4°C should be remain on room temperature before start. 3) Clean the Hood with 70% ethyl alcohol. 4) Then keep all necessary things in culture hood after rinsing them with alcohol. 5) Discard should be there in hood to discard the spent media of culture flask. 6) Check the confluency of cells first under microscope. Cells should be healthy. 7) Never split the cells which are not confluent. Otherwise it will stress to the cells. Cells are very fragile and sensitive so handle very gently and carefully. 8) Always clean your hands with 70% ethyl alcohol. 9) Trypsin/EDTA which is required for cell splitting should be pre-warmed at 37 °C. Procedure for splitting/passaging of cell line from established primary cell line (Like Vero cell line which is an adherent cell line)
1) Take the confluent flask and remove all the spent media using serological pipette. 2) Wash the monolayer of cells once with Phosphate buffer saline. 3) Then take 1 mL of Trypsin/EDTA solution and let it cover the monolayer completely. 4) Remove some trypsin solution. 5) Keep the flask at 37°C in BOD incubator for approx. one and half minutes. 6) Tap gently on the side of flask so that cells can dislodge. 7) Add about 2-3 mL of 10% complete DMEM media to flush off the cells that have got dislodged. 8) Centrifuge the tube containing the cells at 1500 rpm for 5 minutes. 9) Cell pellet will form. 10) Now dissolve the pellet in 2 ml fresh DEMEM media. 11) Count the cells by hemocytometer and take the cells accordingly into the new flask to continue the cell line. 12) Check the flask after 24 hour for its quality and any contamination. 13) Split this flask at alternate day to maintain the cell line for long time (i.e. upto 30 passages). How freezing of cells in done in Liquid nitrogen
• After trypsinisation of Confluent culture flask , forms pellet and dissolve in DMEM media(as mentioned in previous slide). • Count the cell to get an idea about cell density. • Then add 2-3 million cells per cryovial on freezing media. • Freezing media is basically DMEM media with 10% FBS and 10% DMSO). DMSO is dimethyl sulphoxide which is a cryoprotectant and prevent ice crystal formation. • Keep the cryovial in -80°C and then next day in liquid nitrogen. • In this way our cell line remains with us for a very long time. Source of contamination in cell culture • Contamination of cell line means something which is undesirable in the culture system but comes unknowingly. • Contamination makes our culture media present in cell culture flask fuzzy. Normally it should be clear. • Two reason may be possible for contamination either • 1. Chemical contamination • 2. Biological contamination. • Chemical contamination includes:- ✓ DMEM media ✓ Phosphate buffer ✓ Trypsin/EDTA solution ✓ Incubator • Biological contamination includes:- ✓ Bacteria ✓ Fungi ✓ Mycoplasma (generally observed) ✓ Cross contamination with other cell line (i.e When two cell lines are splitted back to back without giving any UV- radiation, then chances are more). References • Oyeleye et al (2016). Basics of animal cell culture: Foundation for modern science. Biotechnology and Molecular Biology Reviews. Vol. 11(2), pp. 6-16, May 2016. • Yao, T and Asayama, Y. (2016). Animal-cell culture media: History, characteristics, and current issues. Reprod Med Biol. 2017;16:99.