Standardized Manipulation Of Human Feeder Preparation

People are attracted by human embryonic stem cells (hESCs) sincetheir discovering, as they have the characters of self-renewing andploripotent abilities. Any kind of hESCs research is based on the premisethat we can get hESCs steadily in a long term. Co-culture with mouseembryonic fibroblast Feeder(mouse Feeder) is the earliest way to gethESCs, immediately after that human embryonic fibroblast Feeder(human Feeder) takes into use, scientists culture hESCs so long time thatthe stability of these two methods can be well approved. However, themost widely used Feeder which is made by mouse embryonic fibroblastsdo not commit the demandings of clinic, to accomplish the goals ofhESCs' clinical transplant we should do our best to avoid the use of anyanimal materials, especially,animal cells. In these years, some labs foundthat there is a nother way to culture hESCs besides co-culture with othercells, it is called Feeder free culture system, but it is still not sure aboutthat whether this way can keep hESCs stable with normal karyotypes. Forthese reasons, co-culture with Feeder which is made by human embryonicfibroblasts comes to be our best choice right now. If we want get largequantities of undifferentiated hESCs, we should firstly get enough humanembryonic fibroblasts and a steady human Feeder-making protocol.We've done some experiments to solve the problems we've ever found inour human Feeder making, and then formulated a new standardFeeder-making protocol.Aims:1. Collect enough human embryonic fibroblasts for Feeder-making;2. Do some experiments to solve the problems we've ever found inour human Feeder making; 3. Formulate a new standard Feeder-making protocol according tothe data we've got from these experiments, so that we'll have aguideline when we are making human Feeder.Materials and methods:We choose the whole fetal body that abandoned in abortion surgeryfrom Changsha Maternal And Child Care Service to do our collection ofhEFs. The first step of collection was to separate skin and muscle fromthe limbs of fetal body mechanismly, then, hEFs was derived throughorganization culturing, and pasaged by T/E. We do the examinations oneach hEFs strain that comes from different fetal bodies: Chlamydia,mycoplasma,bacterial culturing as well as hoechst33258 staining. Andwe also examined karyotype of each hEFs strain. When they were suredabout no affection, they would be pasaged to p7 or p8 and dealed withmite, then made into Feeders. These Feeders would be co-cultured withchHES 20 and chHES 22 to test whether they can support hESCs, theywere growing together in D/F ES medium for 6 days, medium waschanged everyday, after that AKP staining and tra-1-60 staining would bedone to each to test whether the newly gathered hEFs strains can be used.To solve the problems we've ever found in our human Feedermaking, we'd done following experiments:1. We made Feeders with different cell densities by hEF-dly-2 p7,co-cultured them together with chHES 20 and chHES 22 for 6 days, thenstained these clones by AKP solution to see whether there weredifferences between each density groups; we used hi-DMEM as a basicmedium- added SSS, HSA, FBS for each goup,and cultured hEF-E-1 p7cells- to fred whether there are some other subtitudes of FBS can be usedto proliferate hEFs efficiently;2. During in vitro culture of hEF, we compared the differentiated rates of chHES 20 and chHES 22 cultured on Feeder which were made indifferent phase of growth curve(lag phase, exponential phase, plateauphase, decline phase);3. We made Feeders with different cell densities by hEF-dly-2 p7,co-cultured them together with chHES 20 and chHES 22 for 6 days, thenstained these clones by AKP solution to see whether there weredifferences between each density groups;4. We compared the differentiated rates of chHES 20 and chHES 22which were cultured on Feeders that made by 3 different hEFs (mitehEF-dly-2 p7, mite hEF-dly-8 p7, mite hEF-dly-9 p7);We got datas for formulated a new standard Feeder-making protocolfrom these experiments.Results:We've totally collected 10 hEF strains, 9 of them can prolifertatenormally, and all of these 9 strains are without Chlamydia, mycoplasma,bacterial affections, all of these 9 strains can support chHES 20 andchHES 22;1. When compared about the proliferates between FBS,HAS,SSSgroups, we've found that 10%FBS group is still the best one;2. The best result comes up when we use the hEF-dly-2 p7 thatcultured until they have reached the tail of exponential phase afterpassaging, the hESCs' undifferentiated rate is 1.7% only;3. To cope with the hESCs culture system of D/F ES, human Feedershould be made at a density of 3×10~4个/cm~2;4. There is no difference at differentiated rates among thesame-strain hESCs if we culture them on different-strain humanFeeders;We've formulate a new standard Feeder-making protocol. Conclusions::We've got 9 hEF strains that commit the demands of making humanFeeder. If there is no negative effect to cell life by freezing, each hEFstrain can be used at our lab's consumption scale for at least 26 years.1. We've not found a suitable substitute for FBS yet, but instead, wecan wash the Feeder with phosphate-buffered saline (PBS) to removeFBS。2. hEF-dly-2 p7 needs 10 days to cover lag phase(1 day),exponential phase (5 day), plateau phase (4 days) and then reach declinephase, when passaged at 1: 20. The data indicates that the best one ismade up by hEF-dly-2 p7 cells when they cultured until the end ofplateau phase.3. the density of human Feeder cells has obvious effects to hESCsunder the D/F ES culture system while it will leads to seriousdifferentiation of hESCs, no matter lower or higher.4. there is almost no difference among the differentiate rates ofhESCs that co-cultured with Feeders made from 3 different hEF cell lines.It suggested that our methods are stable.We used this new standard Feeder-making protocol for a guide inpast one and half years, and 3 hESCs strains were keeping cultured withlow differentiate rates and normal karyotypes.