Xpert TM Animal Tissue Culture Teaching Kit Product Code: CCK005 Contents 1. About the kit 2. Kit contents and storage instructions 3. Materials required but not provided in the kit 4. Aseptic techniques and good cell culture practices 5. Instructions for use 6. Protocols 6.1 Preparation of complete medium 6.2 Thawing of cryopreserved cells 6.3 Sub-culturing of the cells 6.4 Estimation of viability and enumeration of the cells 1. About the kit Xpert TM Animal Tissue Culture Teaching Kit has been developed for teaching basic animal tissue culture techniques in educational organizations, where the sophisticated facilities required for culturing of animal cells may not be available. CCK005, Xpert TM Animal Tissue Culture Teaching Kit is sufficient to perform 20 subcultures post thaw. Long term cryopreservation of cells without loss in viability requires storage in liquid nitrogen atmosphere at -180 o C. Hence to use the kit most effectively, it is recommended to thaw the cells and start the procedure immediately after receipt. In our experience, CHO cells lost viability when stored in the upper chamber (freezing compartment) of a frost-free refrigerator. a. *Chinese Hamster Ovary (CHO) Cells: CHO cells are fibroblast cells which are derived from the ovary of the Chinese hamster. They grow as a monolayer and can be cultured in a CO 2 independent atmosphere. Two vials of CHO cells have been provided so that one vial can be used a back up vial in case of failure to revive or propagate the cells in the other vial. CHO cells if cryopreserved at -180 C have an indefinite shelf life. Shelf life of all other reagents is minimum one year from the date of manufacture. b. Leibovitz s Medium: : Leibovitz s Medium is a growth medium specifically designed to grow cells in a CO 2 free atmosphere. The standard sodium bicarbonate/co 2 buffering system is replaced by a combination of free base amino acids, phosphate buffers and higher levels of galactose and sodium pyruvate so that the medium does not require supplementation with sodium bicarbonate and can be used under conditions of free gaseous exchange with atmosphere. c. Fetal Bovine Serum: Fetal Bovine Serum (FBS) serves as a source of proteins, vitamins, carbohydrates, lipids, hormones, growth factors, minerals and trace elements. FBS contains growth factors which promote cell proliferation and has an antitrypsin activity which helps in cell attachment. d. Antibiotic Antimycotic Solution: Antibiotic Antimycotic solution helps to prevent microbial contamination. This solution is a mixture of Penicillin, Streptomycin and Amphotericin B in 0.9% normal saline. It is effective against Gram positive bacteria, Gram negative bacteria, Fungi and Yeast. e. Trypsin EDTA Solution: Trypsin EDTA Solution is a cell dissociation solution containing Trypsin and EDTA in Dulbecco s Phosphate Buffered *Cells supplied with this kit are strictly for educational activity in teaching institutions and should not be used for any other commercial purpose.
Saline. Trypsin is a serine protease commonly used for dissociation and disaggregation of adherent cells. Ethylenediaminetetraacetic acid (EDTA), a chelating agent is added to enhance enzymatic activity of trypsin solution. EDTA acts by chelating calcium and magnesium ions that enhance cell to cell as well as cell to flask adhesion. 2. f. Dulbecco s Phosphate Buffered Saline (DPBS): DPBS is a balanced mixture of synthetic salts that maintains ph and osmotic pressure in the medium and provides adequate concentration of essential organic ions to the cells. It is used to wash the monolayer of cells as it is free of calcium and magnesium. It does not hinder the trypsin activity. g. Trypan Blue Solution: It is a vital stain. It selectively stains the dead tissues or cells which after staining appear blue in colour. Live cells or tissues with intact cell membrane are selective for the compounds that pass through the membrane as a result of which trypan blue is excluded by viable cells and they remain colourless. h. Tissue Culture Flasks: Tissue Culture Flasks provided in this kit have a vented cap, surface area of 25cm 2 and a working volume of 5ml. These are surface treated flasks which are specifically used to culture adherent cells. 2. Kit contents and storage 7. 8. On receipt, remove the contents of the kit and place them in 9. appropriate storage locations as per recommended storage temperature. 10. Code TCL095 AL011A RM10432 A002 TCL007 TL1006 TCL005 TCG4 Contents Quantity Store at 12. Description Cryopreserved Chinese Hamster Ovary 13. 2 vials -20 C / - Cells 14. Each vial contains 5X10 15. 6 cells/ml 170 C Leibovitz's L-15 Medium 16. 90ml 2-8 o C With L-Glutamine Fetal Bovine Serum 17. 10ml -20 o C 18. Antibiotic Antimycotic Solution 100X w/10,000u Penicillin, 10mg Streptomycin 19. 1ml -20 C and 25µg Amphotericin in 0.9% normal 20. saline Trypsin-EDTA Solution 1X 21. 0.25% Trypsin and 0.02% EDTA in 22. 15ml -20 C Dulbecco's Phosphate Buffered Saline 23. Dulbecco's Phosphate Buffered Saline 1X 24. 25ml 15-30 C Without calcium and magnesium Trypan Blue 0.5% solution in Dulbecco's 25. 10ml 15-30 C Phosphate Buffered Saline 26. Tissue culture flask 25cm 2 27. 10Nos 15-30 C, Surface treated, vented cap 28. 29. 30. 31. For long term storage Quantities supplied in excess to compensate operational losses.
3. Materials required but not provided in the kit 3.1 Equipments Laminar air flow hood Incubator at 37 C Inverted microscope with 10X objective Hemocytometer with cover slip Centrifuge Water bath at 37 C 3.2 Consumables Micropipette Tips (1000µl, 200µl) and tip boxes Serological pipettes 15ml centrifuge tubes 1ml eppendorf tubes Pipette aid (LA692) Disposable gloves Lab coat Isopropanol spray Tissue paper 4. Aseptic techniques and good cell culture practices a. Use Personal Protective Equipment (PPE), (laboratory coat, gloves and eye protection) at all times while working in a cell culture lab. Use head caps to cover hair. b. PPE for tissue culture facility should be kept separate from PPE worn in general laboratory environment. c. Before starting tissue culture work switch on the UV light in the cabinet for 15-20 minutes. d. Keep all the work surfaces free from clutter. e. All reagent and media bottles should be labeled correctly with name and date of preparation and should be kept at recommended storage temperatures. f. Clean the working area of the laminar air flow hood with 70% isopropanol. g. Prior to starting work all reagent and media bottles, pipettes, tip boxes should be sprayed with 70% isopropanol. h. Arrange the work station in such a way that you have an easy access to all the items and a wide clear space in the centre of the bench. i. Keep all the reagents and media bottles to the left hand side of work station and the consumables and discard beaker to the right hand side of work station for efficient working. j. While working do not contaminate the gloves by touching anything outside the cabinet (especially face and hair). In case they become contaminated then, respray with 70% isopropanol before proceeding. k. In case of any spillage while working, mop up immediately and swab the area with 70% isopropanol. l. Avoid rapid movement within and immediately outside the cabinet. Slow movement will allow the air within the cabinet to circulate properly. m. Avoid speaking, sneezing and coughing while working in the cabinet to prevent the contamination. n. Pipette tips, waste reagents and waste medium should be discarded carefully into a separate discard beaker. o. Once the work is finished, clear the working area and clean with 70% isopropanol.
5. Instructions for use Prepare complete medium as per protocol no. 6.1 Thaw the frozen cells as per protocol no. 6.2 Seed the cells in T-25 flask as described in protocol no. 6.2, step (f) After 2hrs Check for cell attachment Cells attached Cells not attached Incubate the cells for another two hours Give medium change as described in protocol no. 6.2, step (j) Incubate at 37 o C Flask will be 80 90% confluent Sub-culture the cells as per protocol no 6.3 Estimate the viability & enumerate the cells as per protocol no 6.4 Seed the cells in T-25 flask for further maintenance as per protocol no 6.3.2 Flask will be 80 90% confluent
6. Protocols Read the entire procedure carefully before starting the experiment. 6.1 Preparation of complete medium a) Thaw Antibiotic Antimycotic Solution (A002) and Fetal Bovine Serum (RM1112) by keeping the bottles at room temperature for 30 minutes or in a 37 C water bath. Note: Temperatures higher than 37 C can result in deterioration of reagents. b) Once Antibiotic Antimycotic Solution and FBS are thawed completely, remove the bottle of Leibovitz s Medium (AL011A) from the refrigerator. c) Disinfect all bottles with 70% isopropanol on the outside with a quick spray or wipe with 70% isopropanol before placing them in laminar air flow hood. d) Add 10ml of RM1112 and 1ml of A002 to 90ml of Leibovitz s medium (AL011A) using all precautions. Swirl the bottle to ensure uniform mixing. e) The complete medium is now ready for use. Note: Complete medium should be stored at 2-8 o C. It should be used within one month from the day of preparation. 6.2 Thawing (Revival) of cryopreserved cells Requirement: Frozen vial of cryopreserved cells Complete growth medium Tissue culture flask (T-25) Pipettes Laminar air flow hood Water bath at 37 o C, Incubator at 37 o C 70 % isopropanol Procedure: a) Do not remove the vial of cryopreserved cells from -20 o C until the culture flask is ready as below. b) Warm the complete medium by keeping the bottles at room temperature for 30 minutes or in a 37 C water bath. Note: Temperatures higher than 37 C can result in deterioration of the medium. c) Label one T-25 flask and keep it ready in laminar air flow hood. Label should contain the following information. a. Name of the cell line b. Passage number c. Date d) Aseptically add 5ml of pre-warmed complete medium to the flask. e) Place the frozen vial of cells in a water bath at 37 o C. Hold the frozen vial of cells in the water bath with lower half immersed in water. Keep shaking the vial until the frozen clump inside it thaws completely. Note: Avoid getting the water up to the cap of the ampoule to decrease the chance of contamination. f) Swab the vial thoroughly with 70% isopropanol and open it in a laminar hood. g) Immediately transfer the contents of the ampoule to T-25 flask containing complete growth medium. h) Rock the flask gently to ensure proper mixing of cell suspension and the medium and incubate at 37ºC. Note: Flask should be always placed horizontally in the incubator. Do not incubate the flask in upright position. i) Two hours after incubation, check for the attachment of cells by observing the flask under an inverted microscope. j) Place the flask upright in the laminar air flow hood and carefully aspirate entire medium from the flask using a pipette. This is done to remove the cryoprotectant DMSO. DMSO at frozen temperature protects the cells by preventing formation of ice crystals. However, at room temperature, it is toxic to the cells. k) Add 5ml of complete medium to the flask and further incubate it at 37ºC till it becomes 80 90% confluent. l) Change the medium after every 48 hours. m) After every 24 hours check for the attachment and confluence of the cells by observing the flask under an inverted microscope. Precautions: Do not use incubator or the palm of your hand to thaw the cell cultures since the rate of thawing achieved is too slow resulting in loss of viability. Use water bath as prescribed in the procedure. Thawed cells should be immediately transferred to growth medium.
6.3 Sub-culturing of the cells The process of sub-culturing is divided into two stages: 6.3.1 Dissociation of cells from culture vessel. 6.3.2 Splitting (diluting) the dissociated cells into appropriate ratio and seeding in fresh medium. 6.3.1 Dissociation of cells from culture vessel Requirement: Dulbecco s (TL1006) Phosphate Buffered Saline Trypsin- EDTA solution (TCL007) Complete growth medium Pipettes Laminar air flow hood, Incubator at 37 o C 70 % isopropanol Procedure: a) Examine the culture flask under an inverted microscope carefully for confluency as well as signss of contamination or culture deterioration. Split the flask if it is 80-90% confluent. Note: It is important to examine your cultures daily and always prior to sub-culture. Always split the cells before they reach 100% confluency.. b) Place the flask upright and remove the spent medium from the T-25 flask by aspiration. c) Add 1ml Dulbecco s Phosphate Buffered Saline (TL1006) to the flask. (This step is required to remove the traces of serum and divalent cations like calcium and magnesium which hinder action of trypsin.) d) Rinse the cell sheet by rocking the flask for 1 to 2 minutes so that the whole monolayer is briefly washed with PBS. e) Discard the wash solution by aspiration. f) Add 500µl of Trypsin-EDTA solution (TCL007) to the flask. Note: The volume should be sufficient enough to completely cover the monolayer of the cells. g) Rock the flask to ensure that the dissociation solution covers the cell sheet. h) In addition to rocking gently, flasks of cell lines that are characteristically difficult to removee from substratum may be tapped to expedite removal. i) If necessary, incubate the flask at 37 C for 1 to 2 minutes. Monitor the processs by observing the flask under inverted microscope. When dissociation is complete, cells will be in suspension and appear rounded. Note: The exact time needed to dissociate cells will vary according to the cell line. The dissociation processs should be monitored closely to avoid cell damage. j) Once the cell dissociation is complete, add 5ml of complete medium to the flask to inhibit the tryptic activity which may damage the cells. k) Disperse the cells into a single cell suspensionn by slow, repeated pipetting. l) Estimate the viability and enumerate the cells. (Refer Protocol no. 6.4) Fig a. CHO cell Fig b. CHO cells monolayer before after trypsinization trypsinization Precautions: Do not exposee the cells to Trypsin-EDTA solution for longer time. Prolonged exposure can damage the cells. It is very important to neutralize the trypsin by addition of serum (or complete medium in this case) prior to seeding the cells into the flask for proper attachment of cells to the flask. 6.3.2 Splitting the dissociated cells into appropriate ratio and seeding in fresh medium Requirement: Cell suspension with a known concentration Complete growth medium Pipettes Laminar air flow hood, Incubator at 37 o C 70 % isopropanol Procedure: a) Determine the concentration of the trypsinized cell suspension as per protocol no. 6.4.2 b) Using the following formula, calculate the amount of cell suspension that should be added to the new T-25 flask to get a required cell density. Formula: C1xV11 = C2xV2 Therefore, V1 = C2xV2 C1
Where, C1 = Cell concentration obtained per ml C2 = Required cell concentration per ml V1 = Volume of cell suspensionn to be added V2 = Total volume of seeding Example, C1 = 0.5 x10 6 cells per ml C2 = 0.1 x10 6 cells per ml V2 = 5ml Then, V1 = 0.1 x10 6 cells per ml x 5ml 0.5 x10 6 cells per ml = 1ml Therefore, 1ml of the cell suspension should be added to the T-25 flask to obtain the required cell density of 0.1 x10 6 cells per ml. c) Add the required amount of cell suspension (as calculated in step no. 2) to the T-25 flask. d) Add the completee medium to the T-25-Flask while maintaining the total volume of the seeding. (i.e. as per the example add 4ml of complete medium to maintain the total volume of 5ml) e) Incubate the flask at 37 C and observe after 24 hours. 6.4 Estimation of viability and enumeration of cells Requirement: Trypsinized cell suspensionn Trypan Blue solution (TCL005) Hemocytometer with coverslip Inverted Microscope with 10X objectivee Pipettes Tips Procedure: a) Mix the dissociated cell suspension in the T- 25 flask thoroughly by pipetting. b) Take 50µl of this suspension into an eppendorf tube. c) Add 50µl of Trypan Blue solution (TCL005) in 1: 1 proportion to it and mix it properly by pipetting. d) Prepare the hemocytometer by gently cleaning the slide surface with 70% isopropanol taking care not to scratch the semi silvered surface. Place the cover slip over the hemocytometer counting chamber. e) Using a pipette place a drop of cell suspension at the edgee of the chamber. The cell suspension when expelled from the tip will be drawn under the cover slip by capillary action. Note: Do not leave the slide for more than 1-2 minutes, as viable cells may die and begin to take up the stain. f) Place the slide on the microscope and using a 10X objective, count the total number of cells (stained as well as unstained cells) and number of deadd cells (stained cells) from all the four WBC chambers. Stained (Blue colored) dead cell Unstained (colorless) viable cell 6.4.1 Estimation of viability of cells Calculate the percentage of viable (unstained) cells as per the following formula. Formula: Percentage of viable = No. of viable cells X 100 cells Total no. of cells Note: To get accurate percentage viability, each viability assay should be performed in triplicates, followed by taking an average of all the three readings. 6.4.2 Enumeration of the cells Calculate the concentration of cells per ml as per the following formula. Formula: C = A x D x 10 4 Where, C = Concentration of cells per ml A = Average number of cells D = Dilution factor i.e. 2 (1:1 dilution) 10 4 = Conversion factor for counting chamber Counting chamber
Example: If total count in four chambers is 100, Then, A= 100/4 = 25 and D = 2 C = 25x2x10 4 = 50 x 10 4 = 0.5 x10 6 cells per ml Precautions: Trypan blue is toxic and a potential carcinogen. Avoid direct contact with skin and eyes. The following points should be considered while loading the cell suspension onto a hemocytometer chamber. 1. Avoid getting air bubbles while loading the cell suspension. 2. Do not overfill the chamber as this will cause the sample to run into the other chamber. 3. Incompletely filled chamber will result in uneven distribution of cells. Revision No. 0/2011-07 Disclaimer: User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and other related HiMedia Publications. The information contained in this publication is based on our research and development work and is to the best of our knowledge true and accurate. HiMedia Laboratories Pvt Ltd reserves the right to make changes to specifications and information related to the products at any time. Products are not intended for human or animal diagnostic or therapeutic use but for laboratory, research or further manufacturing use only, unless otherwise specified. Statements contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents. HiMedia Laboratories Pvt. Ltd. A 516,Swastik Disa Business Park, Via Vadhani Ind. Est., LBS Marg, Mumbai 400086, India. Customer care No.: 022 6147 1919 Email: info@himedialabs.com