The overall goal of the following experiment is to describe how to mass produce human pluripotent stem cells using a simple and cost-effective gelatin coating. This is achieved by first isolating the chorionic VII from human placenta tissues, and then mincing the villa into small tissue pieces as a second step. The tissue pieces are cultured on gelatin coated plates where they form colonies of adherent fibroblasts like cells in five to seven days, and are then used to make conditioned media for human pluripotent stem cell culture.
Next 80 to 100 clumps of human pluripotent stem cells are seeded covered with human placenta derived cell conditioned, medium, and subculture once every four to five days. The results show similar differentiation and attachment of cells grown using the gold standard conditions compared with a method described here of cells grown on gelatin coated plates in human placenta conditioned media. The main advantage of this technique over accessing method is that it use components secreted from human cells without requiring additional exogenous supplementation and synthetic soft trait.
To begin coat T 25 culture dishes with five milliliters of a 0.1%gelatin solution for one hour at 37 degrees Celsius, then sterilize 500 milliliters of phosphate buffered saline, a pair of forceps, surgical scissors, micro centrifuge tubes, and gauze by autoclaving them at 121 degrees Celsius under 15 pounds PSI for more than 15 minutes. Next, prewarm sterile filtered DMEM containing 20%fetal bovine serum. 100 units per milliliter of penicillin and 100 grams per milliliter.
Streptomycin obtain surgically separated human placenta chorionic plates collected from healthy pregnant women at seven to 32 weeks of gestation. After obtaining written informed consent, then wash the plates with five milliliters of PBS using a pair of scissors. Isolate the chorionic VII from the human placenta chorionic plates, and mince the tissue into tiny pieces.
Then wash the tissue pieces three times using one milliliter of PBS for each rinse, place the minced VII into a micro centrifuge tube and use a pipette to wash the samples with 500 microliters of PBS by gently pipetting up and down. Then centrifuge the tube at 120 Gs for three minutes to pull down the minced vi eye. Wash the tissue twice more with 500 microliters of prewarm culture medium, repeating the centrifuge.
Step in between each wash culture, the mince tissue, and medium on the previously prepared gelatin coated plates at 37 degrees Celsius and 5%carbon dioxide. After approximately five to seven days of culture in the same medium, the formation of fibroblasts like colonies undergoing adherent growth can be observed after seven days. In culture exchange.
The media during culture removing any floating cell debris as the growing placental fibroblast will attach to the plate, continue to exchange the medium every two to three days until the third passage culture. The fibroblast like cells derived from the human placenta chorionic plates up to the 10th passage. Then harvest them with trypsin to be used as stock samples for human pluripotent stem cell cultures after excluding contamination as described in the accompanying text protocol at one times 10 to the seventh cells in a T 75 flak culture plate and culture, the cells at 37 degrees Celsius and 5%carbon dioxide until they are 85 to 90%confluent.
Treat the attached cells with 10 micrograms per milliliter of mitomycin C for two and a half hours, and then wash the cells three times with 10 milliliters of prewarm PBS. Next, incubate the cells in prewarm medium without mitomycin C for 24 hours one day after treatment. Replace the media on the cells with 10 milliliters of Dmf.
12 media supplemented with 20%of a serum replacement, 0.1 millimolar beta mercaptoethanol, 1%NEAA, and 1%penicillin streptomycin. After 24 hours of incubation, harvest the condition medium in a 15 milliliter conical tube and filter it using a 0.22 micron syringe filter. Then add another 10 milliliters of fresh medium to the cells and continue to incubate them at 37 degrees Celsius and 5%carbon dioxide.
Collect all supernatants from the cultures every day for a week and freeze the harvested medium at negative 80 degrees Celsius. Freeze the medium in a new container each time. To avoid repeated freeze thaw cycles, obtain a human embryonic stem cell line such as the H one line and begin culturing them by following the manufacturer's instructions for the experimental groups culture.
The same cells on 0.1%gelatin coated plates in placenta derived cell culture. Medium, perform routine packaging once every five days using standard mechanical based packaging methods. Passage the control cells one to four, and the experimental group between one to six and one to 10.
Replace the medium with fresh, medium daily. Beginning at day two. Human pluripotent stem cell lines can be maintained on 0.1%Gelatin coated plates in human placental derived cell culture, medium at a level similar to that observed for cells maintained in typical stem cell culture.
Medium on basement membrane matrix coated plates to confirm that the human placental derived cell culture medium accessed other matrices. The stem cells were subculture on laminin vitro nin and non coated dishes. The cells attached and grew on the laminate and vitro nin coated dishes.
However, after several passages, many colonies were differentiated. Unlike those on the gelatin coated group. After transfer to the non coated dishes, no attachment was observed for stem cells.
The H one and IPSE cell lines shown here were cultured for 10 passages under experimental conditions. After two weeks, they formed embryo bodies that contained mesoderm, ectoderm and endoderm as measured by immunofluorescent staining After its development. This technique paved a way for researchers in the field of human stem cell biology to explore clinical applications.
Since this method, ovate non contamination with by animal product.
