The overall goal of this procedure is to perform a cell transplantation using genetically modified injectable steroids. This is accomplished by first preparing 3D cell OIDs on micro patterned culture plates using rat hepatocytes or MSCs. Next, polymers and plasmid DNA solution are thoroughly mixed to prepare plex nano my cells.
Then the PHE are transfected by adding the plex nmy cell solution to the culture medium. Finally, the steroids are recovered by placing the plates on ice and aspirating the detached steroids. Ultimately, the genetically modified steroid in suspension are used for cell transplantation to achieve prolonged transgene expression from the transplanted cells in host animals, while maintaining the innate function of the cells, Demonstrating the procedures by Ms.Ula, Ms.Yha, Ms.Suzuki, Mr.Matsui and Dr.They are all in our laboratories.
To isolate hepatocytes, use a special solution outlined in the text protocol to perfuse the livers of spra dolly male rats from the portal vein. Follow the perfusion by circulating a collagenase solution through the liver. After the perfusion, carefully remove the liver and use a scalpel blade to gently mince it.
Then add culture medium and filter the cell suspension through a 100 micrometer nylon mesh to remove additional debris. Centrifuge the cell suspension at a low speed such as 20 Gs for one minute. Then transfer the supernat containing the hepatocytes to a fresh tube centrifuge again and repeat for a total of three times.
Centrifuge the hepatocytes at a high speed, such as 50 Gs for three minutes to pellet the cells and use the special culture medium outlined in the text protocol to resuspend the cells at a concentration of four times 10 to the fifth cells per milliliter to obtain rat MSCs. After resecting the femurs and tibia, collect the bone marrow by inserting a 22 gauge needle attached to a syringe filled with 10 milliliters of culture medium into the shaft of the bone and flushing it out. Seed the hepatocytes or MSCs on 12 well micro pattern plates at a density of four times 10 to the fifth cells per well and incubate them at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide.
The cells will accumulate on the micro pattern adhesion areas and gradually form round steroids within two days. To prepare plex nano my cells after synthesizing the peg PASP debt block co-polymer, and the PASP debt homo polymer outlined in the text protocol, make a solution containing an equal molar ratio of mixed residual amino groups in 10 millimolar heaps buffer by adjusting the polymer concentration to 33.3 micrograms per milliliter and 19.1 micrograms per milliliter respectively. Then add the pre-mixed solution of the two polymers to the PDNA solution at a volume ratio of two to one and thoroughly mix them.
After incubating the cells for 72 hours to allow the formation of mature spheres to carry out gene transfection, replace the culture medium with one milliliter of fresh medium and add 100 microliters of the plex anom cell solution to each well incubate for 24 hours. Then to recover the cell spheres, replace the culture medium with 200 microliters of chilled PBS and place the plates on ice. Use a syringe with a 23 or 27 gauge needle to gently aspirate the cells for in vivo injections.
The transgene expression in the sphe is confirmed by in vitro Gaussian luciferase transfection. The luciferase secreted in the culture. Medium is estimated using a commercial renal, a luciferase assay system and a luminometer after collecting 50 to 100 microliters of culture medium for the in vivo evaluation of the transgene expression in host animals following cell transplantation after anesthetizing.B-A-L-B-C.
Nude mice, according to the text protocol, inject 200 microliters of the cell suspension containing SPS transfected with GL four luciferase expressing PDNA into the subcutaneous tissue of the mouse abdominal region. After injecting d Lucifer, use an IVIS imaging system to measure the luciferase expression to evaluate the therapeutic effects of the cell transplantation. Inject 200 microliters of the cell suspension containing PHE transfected with the erythropoietin encoding PDNA into the subcutaneous tissue of the abdominal region.
Collect blood samples by submandibular bleeding to obtain approximately 100 microliters of blood. Use a blood sample analyzer to measure the hemoglobin and hematocrit in the sample following gene transfection of the Gaia luciferase expressing PDNA using plex nano my cells or the control lipid-based transfection reagent. The nano my cells induced almost no change in steroid structure compared with non transfected OIDs, whereas the control reagent significantly disrupted the structure a day after transfection after transfection using the nmy cells.
Consistent albumin secretion and transgene expression were well maintained for almost a month. The control reagent demonstrated a similar degree of transgene expression, however, no albumin secretion was observed after spheroid recovery from the cooled thermo sensitive micro pattern plates. The 3D structure of the steroids was well preserved for both the primary hepatocytes and the MSCs in suspension as seen here.
After subcutaneous injections of the recovered hepatocyte, PHE Luciferase expression was detected in the animals for more than a few weeks. Albumin expression from transplanted hepatocytes was observed in the host tissue suggesting that the cell function was preserved through the process of spheroid recovery and transplantation. After subcutaneous transplantation of the hepatocytes steroid expressing erythropoietin, a significant hematopoietic effect was obtained in the animals for one month, suggesting the potential for steroids in therapeutic applications.
After watching this video, I usually have a good understanding of how to perform a cell plantation with genetic modification using cells, failed culture systems of thumb sensitive micro pattern plates with gene introduction using narms.
