Experiments in the culture and preparation of samples of pregnancy products and other solid tissue sources for chromosome analysis
Experiments in the culture and preparation of samples of pregnancy products and other solid tissue sources for chromosome analysis
In clinical practice, chromosomal studies require the use of solid tissues other than tumor tissue for one of two reasons: some patients can only obtain solid tissue samples by tissue biopsy, or because of suspected chimerism, tissues are taken instead of standard peripheral blood lymphocytes.
Operation method
Instrumental fragmentation and incubation of tissue samples for mid-division chromosome analysis
Materials and Instruments
Tissue samples Move basic program 1. Place the tissue specimen into a 35 mm Petri dish containing 5 ml of HBSS and 5 times the concentration of antibiotics using sterile dissection instruments. Leave at room temperature for >30 min. 2. Transfer the tissue specimen to a new Petri dish containing approximately 2 ml complete medium/FBS (supplemental medium, e.g. Chang's Medium, Unit 8.2 or IrvineScientific, may accelerate growth). Shred the tissue into fine pieces with fine dissecting scissors and fine forceps or with a scalpel. 3. Transfer the tissue fragments and as little Complete Medium/FBS as possible (approximately Iml per flask) into 2 or 3 25 cm2 plastic tissue culture flasks using a sterile pasteurized pipette, distributing the tissue fragments evenly throughout the bottom of the flasks (widest side). 4. Carefully place the culture flasks in an upright position (bottom side vertical, lid facing up), screw the lids on tightly and incubate for several hours or overnight. 5. Add 4 ml of complete medium/FBS from the opposite side of the tissue cell side in the culture flask, handle carefully so as not to stir up the walled tissue fragments, tighten the cap (to prevent contamination), place the flask flat (so that the walled side of the tissue remains horizontal), and incubate for 2d. 6. Check whether there is any contamination of the plant. Turbidity of the culture medium, acidity of the culture medium, or obvious fungal growth are all indicative of contamination. If there is no sign of contamination, loosen the cap of the culture bottle and continue to culture. 7. After 5d (up to 3 weeks), check the growth of the plant. If a small number of cells, usually fibroblasts, can be seen, they grow around the tissue fragments. Cells from amniotic membrane or some other tissue cultures may be epithelial cells rather than fibroblasts as would normally be the case. These cells, including fibroblasts, are not trypsin-resistant and should be harvested as early as possible if chromosomal smears are to be prepared. 8. Change the medium twice a week while the cells are growing. If possible^§ rinse off large tissue grafts, and when several clones are well grown and have entered the exponential growth phase (e.g. mitotic cells are round and free, with sufficient interclonal space), harvest the cells directly from the primary culture and prepare mid-division chromosome smears as described for amniotic fluid in culture flasks (Module 8.2). The chromosome smears are stained (Module 4.3) and analyzed for karyotype (Appendix 3K). 1. Place the tissues to be examined in separate 35 mm Petri dishes (one dish for each tissue). Add Iml collagenase solution and digest for lh. 2. Using a pipette or syringe, transfer the collagenase solution containing the loosened cells to a 15 ml sterile centrifuge tube. Cover the tube and leave at room temperature. Add Iml trypsin/EDTA solution to the remaining tissue in the dish and digest for lh. 3. Blow the liquid in the dish up and down with a syringe attached to a 20 G needle or a fine pasteurized pipette to break up any remaining clusters of cells. Transfer the cell suspension to the centrifuge tube containing the cell suspension in step 2. 4. Add complete medium/FBS to 12 ml. Centrifuge at 70 g for 8 min at room temperature, discard the supernatant, and resuspend the cell precipitate in 1?2 ml complete medium/FBS. 5. Drop the cell suspension onto 4 coverslips or divide into two 25 cm2 flasks according to the amniotic fluid culture method, and check for contamination after Id. For pre-harvest nutrient cultures, prepare mid-division chromosome smears according to the in situ or flask culture method (Module 8.2). Chromosome smears are stained and analyzed for karyotype. 1. Transport the pregnancy product specimen in a sterile or clean airtight container. 2. If the specimen is to be processed immediately, there is no need to add additional fluids as the specimen already contains enough fluid to maintain its moistness. If the specimen cannot be processed immediately, it should be processed within 24 h. The specimen should not be stored at 4°C for more than 5 d. The specimen should be stored in a closed container. Do not add additional fluids and do not freeze the specimen. 3a. For larger, intact specimens, if the fetal portion is readily identifiable, e.g., whole or large fragmented embryos, carefully remove the fetal portion of the specimen from the container and place it in an appropriately sized sterile plastic petri dish or stainless steel container. Careful search should also be made for fragments of placenta and fetal membranes. Note: Although most samples are not sterilized, so true sterilization is not possible, all petri dishes and equipment should be cleaned and sterilized with alcohol. 3b. For smaller or partial specimens, the entire sample is placed directly in a larger petri dish. The specimen is carefully separated with a dissecting needle or fine forceps, looking for small embryos or embryo parts and fragments of placenta, membranes and armbands. Most of the specimen is probably maternal meconium; meconium tissue does not reflect the genotype of the fetus and therefore cannot be used. The _^appearance_^ of the meconium is membranous and similar to the fetal membranes (e.g., amnion vs. chorionic villus), but the meconium is very brittle. Suspected fragments of placenta or embryonic tissue can be viewed with a dissecting microscope if needed to determine their origin. The placenta has mutilated hairs that can distinguish it from the meconium. In earlier, intact specimens, it is possible that the embryonic tissue may be completely encapsulated within the villi and/or meconium' and the specimen will need to be cut open to look for embryonic tissue. In some incomplete specimens it may not be possible to find embryonic tissue. 4. If the specimen is a fresh, unsoftened fetus, 2 to 3 mm3 of muscle tissue is collected from the extremities for culture. If it is necessary to obtain tissue from other organs to prevent possible chimerism, tissue from the skin, kidneys, lungs, and/or liver can be used for culture. If the specimen is a small, fresh embryo, a portion of 2 to 3 mm3 of the entire sample is examined or photographed at one time. If the embryo or fetus has softened, a 2-3 mm3 fragment of fetal membranes or domain hairs is taken. In many spontaneous abortions and some induced abortions, the embryo or fetus may have died several days prior to expulsion, which results in a discolored, brittle, and soft (softened) embryo specimen, which usually does not have a sufficient number of viable cells for culture. In such cases, the meconium and chorionic villi are usually more viable. Collect biopsy tissue aseptically. Carefully trim the adipose tissue with sterile fine forceps and fine scissors or a scalpel. Place the biopsy tissue in a sterile centrifuge tube containing enough HBSS or unsupplemented medium to cover the tissue and store at 4°C if needed; do not freeze the specimen. For in vivo cultures of fibroblasts, skin biopsies are usually taken from 2 mm diameter by 2 mm deep skin tissue. Other sources of tissue are available for surgical procedures (fascia or other connective tissues are the easiest to culture); in some special cases, specific tissues are studied (e.g., gonadal tissue biopsies in cases of sex chromosome chimerism). These tissue biopsies require a small slice of about 3 mm3. In the case of autopsies of stillbirths or neonatal deaths, larger portions of tissue can be obtained for culture. If there are no other special considerations, deep muscle tissue is the best choice. This is because deep muscle tissue is less likely to be contaminated than skin tissue and can remain viable for 5 to 7 days after death. The specimen can now be used for the preparation of mid-division chromosome smears in culture. After 3-4d of incubation, the plants should be examined for outgrowth. Chorionic tissue obtained from spontaneous or induced abortions can be used for direct preparation of mid-split chromosome smears. Obtain chorionic villus tissue from a chorionic villus biopsy and prepare a direct mid-split chromosome smear as described in Module 8.1. Stain and karyotype as described in Module 4.3 and Appendix 3K. This method of preparation does not yield a large number of schizogony phases and is not of high quality. However, it does not require a large amount of medium and a long incubation time than the methods described above, and there is no possibility of misdiagnosis due to overgrowth of cells of maternal origin. Chromosome slides prepared by the direct preparation method can also be used for interphase nuclear in situ hybridization to screen for some aneuploidies. For more product details, please visit Aladdin Scientific website.
HBSS solution with standard antibiotics
Complete culture medium Petri dishes Dissecting instruments Plastic tissue culture flasks