Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

The overall goal of the following experiment is to reproducibly set up a scalable pluripotent cell culture. This is achieved by first using a pre-programmed liquid handling robot to seed human pluripotent stem cells at varying densities. In a 96 well plate in the second step, the wells with optimal colony densities are visually identified for packaging.

The induced human pluripotent stem cells are then seeded onto new plates for automatic feeding and further packaging. Ultimately, this procedure allows the selection of the best seeding densities for multiplying human induced pluripotent stem cells for their eventual differentiation into cardiomyocytes. The implication of this technique extend towards the development of personalized and regenerative therapies as the medium scale.

URI potent culture robot is suitable for producing a few hundred million URI potent stem cells in several days, which can then be differentiated into various cells for personalized drug testing or cell and tissue implant therapy. Although this method has been applied to screening seeding densities for optimal pluripotent cell culture expansion, it can also be used for optimizing and identifying novel differentiation protocols for various other cell types. Demonstrating the procedure will be pharmaceutical scientists, Neil Daily from my lab Before beginning the feeding procedure, check the 96 swell stem cell colony plate for contamination and to confirm that the cell colony is below the ideal passage density.

If the cells are in good condition, place the plate in bed. Position three on the sloped 37 degrees Celsius ramp of the liquid handling robot and load the tip rack in bed. Position one with room temperature, 200 microliter pipette tips.

Place a four well trough in bed position two and fill well three of the trough with eight milliliters of fresh room temperature, stem cell growth medium without RO kinase inhibitor. Then transfer the platelet to the platelet holding rack and using the robotic control touch pad sequentially select Run the protocol colony feed one plate, and next, then press test run to use the step-by-step wizard to pretest the selected program. When the protocol is approved, select run protocol After the feeding, recover the 96 well stem cell colony plate.

Then record the feeding event details on the plate cover and return it to the cell culture incubator for 24 hours until the next feeding or passage. Choosing the well to split depends on colony density. Several days after a seeding density gradient was set up on this plate, we see a well that is too dense to split an ideal density for splitting and a well too sparse to passage after several feeding cycles.

Confirm that the space between the majority of colonies in the 96 well plate is 25%of the adjacent colony diameter, or a subsequent feeding would result in the colonies growing into one another. Then if the colony is not contaminated, place the plate in bed. Position three on the 37 degree ramp and load the tip rack in bed.

Position one with room temperature, 200 microliter pipette tips. Place a four well trough in bed position two, then leaving the well four trough empty dispense 8.5 milliliters of stem cell growth. Medium supplemented with the row kinase inhibitor Y 2 7 6 3 2 into well three 3.5 milliliters of 30%proteolytic and collagen lytic dissociation reagent into well two and 4.5 milliliters of PBS into well.

One next place, a new prewarm, 37 degrees Celsius extracellular matrix gel coated 96 well plate in bed, position five and transfer the old and new plate lids into the plate lid holder rack. Now use the robotic control touch pad to sequentially select run a protocol colony split one to 12 column one, followed by next. Then select test run to use the step-by-step wizard to pretest a selected program followed by run protocol.

When the passage protocol is finished, recover both plates. Label the new plate with the relevant information and return the plates to the incubator. For 24 hours, the cells and colonies should attach to the plate within two to three hours of splitting, appearing very spread out due to the presence of the RO kinase inhibitor.

After the first RO kinase inhibitor free feeding, the cells will condense and exhibit the characteristic cobblestone stem cell morphology. Chromosomal abnormalities are commonly observed during stem cell culture, but may not affect the distribution of pluripotency markers like those shown in these images is indeed karyotype analysis of cells cultured by the just demonstrated protocol revealed that both the robotically passage inducible pluripotent stem cell lines had 46 normal chromosomes, suggesting that the robotic culture method did not introduce the chromosomal instability beyond what might normally occur. To probe established stem cell gene expression markers in robotically cultured inducible pluripotent stem cell lines, the total RNA was also collected.

Both of the inducible pluripotent stem cell lines in this experiment exhibited a similar expression of pluripotency markers, whereas the cardiomyocytes differentiated from each line did not, nor did a separate line of human dermal fibroblasts. Further, when both inducible pluripotent stem cell lines were differentiated into cardiomyocytes, they exhibited a clear sarcomere formation. Together, these data suggest that three months and more than 20 passages of robotic culture resulted in chromosomally normal cells exhibiting a transcription program consistent with pluripotency that were also capable of differentiation into cardiomyocytes.

After watching this video, you should have a good understanding of how to use this or a similar liquid handling robot to culture pluripotent cells in a scalable manner.