organ culture experiment

Summary

Since the desired organ culture technique involves placing the tissue in a position ideal for gas and nutrient exchange, most of these techniques involve placing the tissue on a hemi-mesenchymal agar gel substrate or on a gas-liquid interface of coagulated plasma, or on a raft of microporous filter membranes, microscope paper, or synthetic fibers supported by stainless steel mesh. Most of these techniques involve placing the tissue on a hemidesmosomal agar gel substrate or at the gas-liquid interface of a coagulated plasma, or on a raft of microporous filters, microscope paper, or artificial fibers supported by stainless steel mesh, or on a gas-liquid interface of strips of organic or resinous glass adhered to a glass strip. This geometry is currently most easily obtained with filter membrane Petri dishes. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition

Operation method

Program 25.1 Organ Culture Experiment

Principle

Remove the organ or tissue and cut it into small 1 mm3 pieces or into membrane or rod shapes. Then, place the tissue on a support located at the air-liquid interface, such as a filter film petri dish. Incubate in a humidified CO2 incubator, changing the culture medium as needed.

Materials and Instruments

M199
Anatomical instruments Filtered Petri dishes 12-well culture plates Fertilized eggs

Move

I. Materials

aseptic

1. Anatomical instruments

2. culture medium with or without serum (e.g., M199)

3. non-tissue culture treated filter membrane petri dishes (e.g., Costar Transwell Polycarbonate, #3423, Corning)

4. 12-well culture plates (Corning)

Non-sterile

1. Fertilized eggs, incubated for 8 d

II. Procedure

1. Place the filter membrane petri dish in each well of the multi-well culture plate and add culture solution until it reaches the level of the bottom of the membrane (about 1 ml).

2. Place the plate in a humidified 37°C constant temperature _CO2 incubator to adjust the pH of the culture solution.

3. Prepare tissue or cut out whole embryonic organs (e.g., femur or tibia of an 8-d chick embryo; see Scheme 12.2 and Scheme 12.7). At one latitude, the thickness of the tissue must be no more than 1 mm, preferably less (e.g., the tibia of an 8-d embryo may be up to 5 mm in length but only 0.5 to 0.8 mm in diameter; a small piece of skin may be 10 ^mm2 but only 200 μm in thickness; tissues that have to be cut into small pieces should be no larger than 1 mm³, e.g., the liver or the kidney).

4. When dissecting specimens over a short period of time (<1 h), HBSS should be adequate. However, for longer dissections, 50% serum should be added to HBSS buffered to pH 7.4 with HEPES.

5. Remove the multiwell plate from the incubator and carefully transfer the tissue to the filter membrane. This step is best done with a Pasteur pipette, which allows you to aspirate the liquid transferred with the tissue mass at the same time, being careful not to puncture the filter membrane. Be careful not to puncture the membrane. Wet the inside of the pipette with culture fluid before aspiration to prevent the tissue mass from sticking to the pipette wall during aspiration.

6. Check the level of the culture solution to confirm that the tissue is moistened, but not completely submerged. Then, place the culture plate back into the incubator.

7. Check again after 2~4 h to make sure that the culture liquid level remains above the filter membrane and the tissue block, but not deep enough to float the tissue block.

8. Culture the tissue for 1~3 weeks, and change the culture medium every 2~3 d according to the need of the samples.

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