TISSUE CULTURE ON COVERSLIPS

I. Purpose:

A. Skin tissue may be used for chromosome analysis in special cases when the results from peripheral blood are inconclusive, e.g. suspected mosaicism, confirmation of a new chromosome disorder, or special dermatological disorders. Tissue may also be used when blood cultures are not available, such as on stillbirths. 

B. The skin biopsy should be performed by a physician, with scrupulous aseptic technique. The biopsy site is thoroughly washed with an antiseptic soap and then wiped with 70% ethanol, which is allowed to dry before the biopsy is taken. Iodine and Mercurochrome-like antiseptics should not be used. Most physicians prefer to use a local anesthetic; this does not affect the success of the culture. A 4mm punch biopsy should be taken and placed in a vial containing 8 ml of transport media (F-10, Eagle's, MEM, etc.). For cases of stillbirth, the preferred tissues are skin, fascia, kidney, and lung. The tube should be labeled with the patient's name and delivered to the cytogenetics lab without delay. 

II. Culture Procedure:

A. Aseptic technique must be used when setting up the cultures, preferably under a laminar flow hood. For each type of tissue label 1 100 mm petri dish with the patient number, patient name, type of tissue, and date. Label a sterile 15ml conical centrifuge tube with patient's number, name," collagenase", time of day, and date.

B. Cultures are grown in complete Chang/F-10 media. Media should be fresh (less than 4 days old) and be prewarmed and at pH 7.0 - 7.5. All cultures are incubated in a wet, 5% CO2 incubator at 37° C. All new cultures are to be placed in the "alien " incubator and checked the next day for contamination. Once cells are growing and determined to be aseptic, they are transferred to the "tissue culture" incubator. 

C. Using sterile forceps, transfer tissue to the petri dish, a 2mm cubed sample is sufficient. If the sample is large, cut off a section. Add 2.0ml of media to the dish. Using scissors, cut tissue into small bits, use clean strokes and avoid mashing or tearing the tissue. Add 4ml media and transfer 2ml into the centrifuge tube and leave 4ml in the dish. Gently rock the p-dish to distribute the media. Place the p-dish in the incubator for 2 - 3 days for the tissue to adhere to the surface; then add 1ml fresh media. The p-dish will be your back-up if the coverslips don't grow or get contaminated. 

D. Allow the tissue in the centrifuge tube to settle while you prepare and sterile filter the collagenase solution. (The concentration of your collagenase solution can vary depending upon the type and amount of tissue you receive. Placental tissues break up easier and require less concentrated collagenase.). Remove the supernatant from the centrifuge tube with a sterile pipette and discard. Add the filtered working collagenase solution. Incubate for 1 to 3 hours, mixing occasionally, until the tissue is broken up. 

E. When the tissue in collagenase is sufficiently broken up; the solution should be cloudy, label culture dishes. For 1 type of tissue received, set up 8 dishes. For 2 types of tissue received, set 6 on each; for 3 or more types of tissue, set 4 on each. Centrifuge the tissue in collagenase for 6 minutes at 150 X g (900 rpm in Sorval GLC-2B). Remove the supernatant, resuspend the pellet and add 2.5ml of complete Chang/F-10 media. NOTE: If your sample size is large or the tissue dissolves nicely in the collagenase, you may want to add more media to make your tissue suspension more dilute. Plate out with 0.3ml on each of 2 coverslips. Add 0.5ml of media to the centrifuge tube and plate out 2 more coverslips. Continue to do this until all coverslips have been plated out. Carefully place the coverslips in the incubator. Check them on an inverted microscope after 2 hours, if possible. If there is adherence, add 1.5ml of media to each dish and recheck the coverslips. (Sometimes, it is difficult to see through the 0.3ml of sample to get a good estimate of adherence so the dishes need to rechecked). If there is significant adherence or growth, remove this media and add 2ml of fresh media. You may want to save the supernatant and centrifuge it to replate if the original coverslips are too dense. With the remaining tissue suspension in the centrifuge tube, you can either discard or transfer to a labeled flask and save as an additional back-up culture. 

F. If there is no adherence or the sample was set late in the day, incubate overnight. The next day, flood the coverslips with 1.0ml of fresh media and check for growth. If there is sufficient growth, immediately change the media. If there is no growth, return to the incubator. The following day, add 0.5ml of media and check for growth. Check the coverslips each day for growth. Some tissues will grow as individual cells, others will grow as colonies. If cells are too dense, metaphases may not spread well. If tissues grow as dense colonies, it may be best to subculture onto additional coverslips. Your mitotic index will be greater and spreading will improve. 

III. Harvest Procedure, In-Situ:

A. Add 20µl Ethidium Bromide working solution to each dish, incubate for 40 minutes. Add 40µl of colcemid working solution, incubate for another 20 minutes.

B. Ten minutes before end of incubation, start up TECAN harvester so that it will be ready.

C. Harvest using TECAN.

If you need to harvest by hand:

1. Remove media; add 2ml hypotonic for 20 minutes.

2. Remove hypo; add 2ml fresh hypo for 20 minutes.

3. Add 2ml fixative to hypotonic.

4. Remove 2ml of fixative/hypo; add 2ml fixative.

5. Remove all; add 2ml fixative for 15 minutes.

6. Remove all; add 2ml fixative for 15 minutes.

7. Remove all; add 4ml fixative for 15 minutes.

8. Remove all; dry in drying chamber or in a humid environment. 

D. Drying conditions are very important for the proper spreading of metaphases. Using the TECAN dry program, remove fixative from the dish. Using the aspirator, remove almost all the fix, going around the edge of the coverslip. Allow to dry in a humid environment, 55 - 60% humidity. If the coverslip dries too rapidly, all the cells will be trapped in the membranes. If it dries too slowly, the chromosomes will float away from the metaphase.

E. Remove the coverslip from the petri dish, keeping it right-side up. It helps to hold the dish with your thumb and middle finger and bend up the bottom of the dish with your index finger so that the coverslip is lifted up. Gently, wipe off any fixative from the bottom of the coverslip with a tissue. Mount the coverslip on a labeled microscope slide using a drop of mounting media. (If FISH is to be performed on these slides, use super glue to mount the coverslips.)

F. Allow the slides to dry at room temperature for at least 30 minutes. Slides can now be baked and banded.

IV. Solutions:

Colcemid working solution: 10µg/ml Colcemid in Hank's Balanced Salt Solution, store at 4° C.

Complete CHANG/F10 media: 45ml Chang basal media B, 42ml Nutrient Mix F- 10, 5ml Chang supplement A, 8ml Fetal calf serum, 1ml Penicillin/Streptomycin, solution, 1.1ml L-Glutamine solution, store at 4° C.

Ethidium Bromide working solution: 2mg/ml Ethidium Bromide in RPMI-1640, stored in dark.

Fixative: 30ml Methanol and 10ml Glacial Acetic Acid, prepared fresh.

Hypotonic solution: 0.8%NaCitrate/0.075 M KCl: 1.6g NaCitrate, 1.68g Potassium Chloride dissolved in 500ml dH₂O. 

1X Trypsin EDTA solution: 10ml stock trypsin solution (10X trypsin in saline), 0.1g EDTA (disodium salt), dissolved in 490ml Hank's Balanced Salt Solution. Sterile filtered, 50ml aliquots. Store frozen.

Collagenase

Stock solution: add 16.5ml complete Chang/F-10 to a 100mg collagenase bottle, dissolve (6mg/ml). Aliquot into microcentrifuge tubes to store at -20 degrees C for up to 6 months.

Working solution: add 0.5ml to 1.0ml thawed collagenase stock solution to 2.5ml complete Chang/F-10 media. Filter, using a 3ml syringe and an Acrodisc filter, into a sterile 15ml conical centrifuge tube. 

V. Reagents:

Chang media: IRVINE cat# T100-018, liquid and frozen supplement,100ml bottle, store supplement frozen, basal media at 4° C.

Colcemid: GIBCO cat# 15210-040. 10µg/ml in Hank's Balanced Salt Solution, 10ml bottle, store at 4 °C.

Collagenase: SIGMA cat# C-9263, 100mg bottle.

EDTA: SIGMA cat# ED2SS. Ethylenediaminetetraacetic acid, disodium salt, dihydrate, 100g bottle.

Ethidium Bromide: SIGMA cat# E-8751. 2,7-diamino-10-ethyl-9-phenyl- phenanthridinium bromide, 250mg bottle.

Fetal calf serum: HYCLONE,INC. cat# A-1111-D. Defined fetal bovine serum, 100ml bottle, store frozen.

Hank's Balanced Salt Solution: GIBCO cat# 14170-112. Hank's balanced salt solution, without CaCl₂, MgCl₂, MgSO₄, 500ml bottle, Store at 4° C.

Glacial Acetic Acid: FISHER SCIENTIFIC cat# A38-500. Acetic acid, Glacial ACS (Aldehyde free), 500ml bottle.

L-Glutamine: GIBCO cat# 25030-032. L-Glutamine solution 100X (200mM), 20ml bottle, store frozen.

Methanol: FISHER SCIENTIFIC cat# A412-500. Methyl alcohol anhydrous (ACS)(Absolute) Acetone free, 500ml bottle.

Nutrient Mix F-10: GIBCO cat# 81200-040. Nutrient Mixture F-10 (HAM) powdered form, prepared to instructions, 10 x 1 liter package. 

Penicillin/Streptomycin: GIBCO cat# 15140-031. Penicillin/Streptomycin solution, 10,000 units/ml; 10,000µg/ml, 20ml bottle, store frozen.

Potassium Chloride: UW-STORES. cat# 2485 ACS granular, 500g bottle.

RPMI-1640: GIBCO cat# 31800-022 RPMI-1640 powdered form, prepared to instructions, 10 x 1 liter package.

Trypsin Stock Solution(1X): GIBCO cat# 25300-013. Trypsin 2.5% (10X) in saline. Store frozen.

Sodium Citrate: UW-Stores, cat# 2588. Crystal, ACS, Na₃C₆H₅0₇^.2H₂0, 500g bottle.