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这个问题请参照“Manipulating the mouse embryo ” (version III) protocol 5 in chapter 8: De Novo Isolation of ES Cell Lines from Blastocysts. 在这个protocol里，酶法和机械法都提及了，请见下面。
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( E. B2 ~' l* n7 b% E& P 1. Day 0: Flush the embryos from the uterine horns (see Collecting Blastocysts) with either M2 medium or DMEM plus 10% serum and 25 mM HEPES (pH 7.4) and place them individually into 10-mm well tissue culture dishes containing a preformed feeder layer of either STO or MEF and 0.5 ml of ES-DMEM.1 s- {6 S& y( Y- M, d# v
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The first stage of embryo culture can also be performed in microdrops of ES medium without feeder cells incubated under light paraffin oil or on gelatinized four-well plates. After 1-2 days of culture, the embryos hatch from the zona pellucida and attach to the surface of the tissue culture dish. Shortly after attachment, the inner cell mass (ICM) becomes readily distinguishable and grows rapidly over the next 2 days. However, embryos may vary considerably. Daily monitoring is essential, without extensive exposure to room temperature and normal atmosphere, which may trigger differentiation.# k" @- G" W' o% ^5 P4 l
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2. ~Days 5-6: ICM cells have proliferated to form a “blastocyst outgrowth.” Dislodge it from the underlying sheet of trophoblast cells using a finely drawn Pasteur pipette. Clumps that have an outer layer of endoderm have differentiated too much and are less likely to generate ES cells. Wash the clump of cells with two changes of Ca++/Mg++-free PBS. Use a finely drawn pipette to transfer the cells to a microdrop of 0.25% trypsin/0.04% (1 mM) EDTA in PBS or similar buffer under light paraffin oil.5 `6 w8 K N+ Q# m
0 {# ?+ s" }- B1 I e$ z An alternative for this and the next steps is to use a micropipettor to pick the outgrowths and transfer them into 25- to 30-μl trypsin drops in a 96-well plate.
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0 p0 o5 x4 l$ y3 ]% S: M% ]$ C 3. Incubate the microdrop at 37°C for 3-4 minutes. Fill another finely drawn Pasteur pipette with serum-containing medium (the diameter of the pipette end should be no greater than the cell clump). Use the filled pipette to disaggregate the “blastocyst outgrowth” gently into smaller cellular aggregates of three or four cells.
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0 i& T" D5 i' w& x1 C. k2 L Do not attempt to reduce the “blastocyst outgrowth” to a single-cell suspension.- W8 A/ u+ G0 I' A& o
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4. Transfer the disaggregated contents of the microdrop into a fresh 10-mm feeder cell tissue culture well. Inspect the individual cultures daily, but only for a brief time.; L: ~+ ^# L% e3 q7 R
' Y% U- s- f! \. ] 5. ~Day 9: Generally, primary colonies of cells will become readily visible now and will have one of several different morphologies:9 D9 ` R4 A' r/ b" e) i; q4 \
8 p/ _; g* N9 d" G" {9 U i. trophoblast-like cells: In nearly 100% of cases, areas of these cells rapidly become apparent. In addition, colonies are frequently present during the early stages of tissue culture that closely resemble pluripotent stem cells but give rise to exclusively trophoblast-like cells over the next 2-3 days.
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: H* a; }, L/ V* C( v4 h2 V ii. epithelium-like cells: Occasionally, colonies of slow growing cells form discrete patches on the feeder layer. The constituent cells pack together to give a flat pavement, epithelium-like structure, often with a very marked, highly refractile edge.4 x3 O0 s$ p0 Z: ~ M& R
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iii. endoderm-like cells: Areas of rounded, refractile, loosely attached cells grow in a few cultures. These appear to be similar to the endodermal cells that form when stem cells are encouraged to differentiate in culture.( \ E. G: a, @8 c: s9 w& z5 C- _
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iv. ES cell-like cells: These are cells that grow progressively and maintain a stable ES-cell-like phenotype ., I7 [4 j* p1 U8 o, L- m
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6. If clumps with ES cell morphology dominate the culture, start passing the cells by the regular trypsinization used for maintenance (see Passage of Embryonic Stem (ES) Cells). If this is not the case, the ES cell-like clump(s) should be physically separated from the differentiated counterparts as described in Steps 2-5.
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7. ~Days 12-13: Reseed cells that resemble ES cells into wells with feeders./ S9 R$ S+ }0 } ~' @% J( O
s4 q8 B+ \7 G+ \& Z 8. ~Day 14: Inspect wells for ES cell colonies.
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- B0 ?7 v! A7 t; y. d7 F$ [7 N 9. ~Day 16: Subculture wells containing ES cells to generate permanent ES cell lines. When ES cell-like colonies dominate the 10-mm well, the cells should be gradually expanded to 35-mm, through 60-mm, and then 100-mm plates. Newly derived ES cells should be expanded to one or two 100-mm plates before they are frozen for storage (see Freezing and Thawing of Embryonic Stem (ES) Cells Using Cryovials).+ P; L8 O/ {+ M- q: Z ?
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(see Troubleshooting)1 a* a+ f0 {1 z- P
( P& a- G+ m* M) o& B" q3 y7 f 10. The sex and karyotype of all newly established ES cell lines need to be determined. It is preferable to have a male ES cell line because XY ES cells can convert the sex of the host embryo in chimera production, and chimeric males can produce more offspring than females. The transmission of the ES cell genome through the germ line of chimeras depends on euploidy of ES cells. Test newly established ES cells for Mycoplasma and murine antibody production.6 U+ _' ^ l: {
7 F( o% I/ E a9 b1 g o# |% p- Z A simple test for Mycoplasma involves staining cultures with the UV fluorescent dye Hoechst 33258. Mouse antibody production (MAP) testing is probably the most sensitive and specific way to identify murine viral contaminants. It is offered by Charles River Laboratories. A PCR-based alternative is faster and is offered by the University of Missouri.
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发表于 2011-10-17 14:16
