| 1 | STEMCELL Technologies | How to Reprogram Fibroblasts into Human Induced Pluripotent Stem (iPS)... | 21169 | 228 | 15 | 52.4 | neutral | 5:07 | This video presents an easy and reliable method for reprogramming human fiber blasts into induced play potent stem cells using repro RNA OKSGM. We'll be demonstrating how to use repro teaser, neonatal human dermal fiber blasts and the repro RNA OKSGM non-integrating vector system. The reprogramming protocol can be broken down into three steps, transfection, induction, and selection. The repro RNA OKSGM self-replicating vector only requires a single transfection. For transfection, we recommend using our repro RNA transfection reagent. Simply mix the repro RNA OKSGM vector in a tube with a transfection reagent and incubate for five minutes. Take your previously plated somatic cells on a major gel or vitro net and XF coated plate and replace the somatic cell medium with growth medium containing B18R. Add the repro RNA vector and transfection reagent mix, drop wise onto the cells. Gently rock the plate for even distribution. After one day, begin the selection process with growth medium containing pure micein and B18R. Change the medium daily for six days. At seven days post-transfection, replace the medium with repro teaser containing B18R and continue changing the medium daily for the remainder of the induction phase. Over the next several weeks, IPS cell colonies will begin to form. When reprogramming fibroblasts, the cells will change shape, appearing more rounded as the mesenchymal to epithelial transition occurs. Around day 8-9, small epithelial-like cells will begin to appear. Around day 14-16, these cells will have developed into small clusters of tightly packed pre-IPS cell colonies. And by three to four weeks, large colonies with ES cell-like morphology will be present. In addition to fully reprogrammed IPS cell colonies, differentiated colonies and partially reprogrammed colonies may be present in your cultures. It is important to accurately distinguish between fully and partially reprogrammed IPS cell colonies to ensure successful selection and clonal expansion of the selected clones. Fully reprogrammed IPS cell colonies generated with repro RNA and repro teaser will display the morphological characteristics of embryonic stem cell colonies. That is, they should have distinct borders. Cells should be tightly packed with prominent nucleolite and have a high nuclear to cytoplasmic ratio. To isolate the IPS cell colonies, either a pulled glass pipette or a 22 gauge needle is recommended. Colony isolation should be performed using a microscope in sterile conditions. Sometimes, the colonies you want to select may contain differentiated cells or fibroblast overgrowth. These unwanted cell types should be removed from colonies prior to isolation. Drag the needle or pipette around the IPS cell colony to separate the fibroblasts or other unwanted cells from the undifferentiated IPS cells you are selecting. Use of repro teaser will generate cultures with fewer fibroblasts and differentiated cells, minimizing the need for manual removal of these cell types. To then isolate the selected IPS cell colonies, drag the needle across the colony, up and down, making a grid. Lift and collect the colony fragments with a 200 microliter micro pipette and transfer to a new culture dish. The dish should be pre-coded with matrogel or vitro-neckedin XF and contain M-teaser or teaser E8. IPS cell lines generated in repro teaser and then subcultured in M-teaser or teaser E8 typically contain very low levels of differentiated cells as early as passage 2. And are therefore ready for banking and characterization earlier than IPS cell lines generated with other methods. Successful generation of new IPS cell lines should display normal carry-o type, express undifferentiated cell markers, and differentiate to all 3 germ layers using either in vitro assays or a teratoma assay. For more information about repro RNA, repro teaser, and or other products for pluripotent stem cell research, please visit stem cell.com. | ↗ |
| 2 | STEMCELL Technologies | How to Thaw Human iPSC Lines | 2113 | 29 | 3 | 49.7 | neutral | 5:17 | In this video, we will demonstrate how to thaw the frozen aggregates of a human-induced pluripotent stem cell line. Proper thawing and handling are essential for optimal recovery of your frozen cells, and to ensure your cell cultures are set up successfully. Refer to the product information sheet specific to the cell line you're working with. The latest version of the product information sheet can be found on the product page. For this demonstration, we'll be using the SCTI-003A cell line. For detailed lot-specific information on quality testing results and recommended culture conditions, please refer to the lot-specific certificate of analysis. In a biological safety cabinet, let an aliquot of M-T-ser-plus media and coated culture wear, such as a matrigel-coated plate, warm up at room temperature before thawing the cells. Do not warm M-T-ser-plus in a 37-degree Celsius water bath. Begin by taking the vial from the cold storage unit and place it on dry ice when transporting it to the biological safety cabinet. We recommend thawing one vial of cells at a time. Wipe the outside of the vial with 70% ethanol or isopropanol. Twist the cap a quarter turn to relieve internal pressure, then retighten the cap. The cell thawing process should be performed quickly to ensure optimal cell viability and recovery. For consistent thawing and reduced variability, we recommend using the automated THOST-RCFT2, or you can thaw the cells in a 37-degree Celsius water bath for 2-3 minutes by gently shaking the vial. Do not allow the vial to thaw completely. Instead, remove the vial when a small frozen cell pellet remains. Wipe the outside of the vial again with 70% ethanol or isopropanol. Use a 2-milliliter serological pipette to transfer the contents of the cryo vial to a 15-milliliter conical tube. It's advisable to use a 2-milliliter serological pipette instead of a 1-milliliter pipette to minimize breakage of cell aggregates. Next, add 5-7 milliliters of room temperature M-T-ser-plus. Drop-wise to the 15-milliliter tube, gently mixing the cells as the medium is added. Gently flick the tube to help with mixing the cell's suspension. Centrifuge the cell's suspension at 300G for 5 minutes at room temperature. Remove the tube from the centrifuge, then aspirate the supernatant carefully, leaving some media behind while keeping the cell pellet undisturbed. Add 1 milliliter of M-T-ser-plus to the cell pellet. Then, resuspend the cell pellet by gently flicking the tube, avoid pipetting up and down. Ensure the cells remain as aggregates for optimal attachment and recovery. Next, take the matrijole coated plate and aspirate the matrijole solution from each well. Then add 2 milliliters of M-T-ser-plus to each well of the 6-well plate. Aliquat the cell suspension into the coated 6-well plate containing M-T-ser-plus at 6 different densities as outlined in the cell line-specific product information sheet. Gently flick the tube as many times as needed to ensure a uniform cell suspension prior to seating the cells into the wells. Flick the tube again between seating into each well, as the cell aggregates will sink to the bottom of the cell suspension. The repeated flicking allows for an equal distribution of the cell aggregates between the wells. Place the plates in an incubator at 37 degrees Celsius and 5% CO2. Move the plate in several quick, short, back and forth and side-to-side motions to evenly distribute the cell aggregates within each well. Note that uneven distribution of cell aggregates may result in increased differentiation of human IPSCs, so it's important to move the plates sufficiently in quick motions. Do not disturb the plate for 24 hours. Before performing the first medium change, it's good practice to check the plate using the microscope to confirm the cell aggregates have attached to the matrix. Perform medium changes as needed using M-T-ser-plus? Refer to the M-T-ser-plus technical manual for examples of a flexible feeding schedule. Visually assess your cultures on a regular basis to monitor growth and morphology. This will help determine the optimal day to passage the cultures. For additional information on passaging or managing spontaneous differentiation in your cultures, please refer to the product information sheet relevant to the cell line you are using. And for more information, visit our frequently asked questions page about IPSCs on the Stem Cell Technologies website. | ↗ |
| 3 | STEMCELL Technologies | Development and QC of SCTi003-A - A Highly Characterized Human iPSC L... | 926 | 9 | | 46.7 | | 7:11 | Hello and welcome to this informational presentation on the development and QC of the Healthy Control Human IPSC line SCTI003A. My name is Andrew Gaffney, Director of Stemsell Manufacturing and Commercialization at Stemsell Technologies and over the next few minutes I'll be walking through some key points about our highly characterized IPSC line. Now it was essential that our commercial IPSC quality assessments and release criteria were developed based on recommendations from a number of internationally recognised groups. That includes ISCBI's Banking Consensus Publication in 2009, Gates Paper on Clinical Grade IPSCs in 2018 and the guidelines rolled out by the ISCCR Standards Initiative in 2023 for the best practices and quality standards for human stem cell use in research. We took on board all recommendations from ISCR and implemented these guidelines fully into our IPSC development processes. And here they are, this is exactly how we develop and test our IPSC lines at Stemsell Technologies. All of the QC procedures that we use in production are listed in this table who are very open about how we do this and as you can see a number of cell banks are generated for each line including a master, working and commercial cell bank. These banks are approximately three passages apart from each other and QC tests are performed at varying stages of the development process. Now as a side to QC we also recognise that a lack of guidelines for naming PSC lines has led to some confusion in the field. So we decided to use the Standardized Nementlature established by HPSE Reg that unambiguously identifies a registered cell line. As you can see in the sensor of the screen our Healthy Control IPSC line has been named SCTI0038. If you scan the QR code on the left you'll see that this takes you to the official page on the HPSE Reg website for the 3A line and that contains all information relating to donor information, ethics, derivation, among other things. You can also download a copy of our extensive 25 page certificate of analysis from there as well. We've provided heightened evidence that the 3A line is pluripotent and has been donor-consented with the highest of ethical standards. Because of this HPSE Reg officially certified the 3A line showing that a minimal set of ethical and scientific standards have been met. This certificate is essential for certain funding agencies including any PSC research funded by the European Union. Now it's important to note that donor tissue is ethically sourced at SAMSEL and is collected using institutional review board or IRB protocols. In the case of 3A this was derived from PBMC's from a 48 year old female who was clinically undiagnosed donation. Dona characteristics are separated out by those that are self declared such as race and ethnicity and those that are calculated like height, weight and blood type. All of our QC can be broken down into a number of different parts, a selection of which can be seen on the screen now. This includes viability and recovery assays, detection of adventitious agents, identity testing, genomic stability and integrity, whole exome and genome sequencing, assessing the undifferentiated states by marker expression and assessing pluripotency by trial and age differentiation. Now in 2017 Florian Merkel and colleagues reported that whole exome sequencing of hundreds of PSC lines identified numerous mutations in P53. Separately to this, Shinny Yamannaka presented findings of a point mutation in the Tumor Suppressor gene B-Corp that also went on to be further described by group at the Sangre Institute. These findings drew attention to the acquisition of mutations in Tumor Suppressor genes in PSCs, so this is also a key focus for our analysis. We performed whole exome sequencing of the 3A line which allowed for the identification of genomic SNPs and the resulting profile of genetic variants was compared against ClinVAR. The statuses of P53 and B-Corp went on to be interrogated in depth. For P53, seven variants were detected and for B-Corp, there were five, however the good news is that they were all silent. Most importantly, no variants were identified that were previously reported by Merkel, Yamannaka and the Sangre Institute, and no pathogenic or likely pathogenic variants were identified by ClinVAR. I should note that despite focusing largely on QC in this presentation, a large amount of the work that went into commercialising the cell line was to demonstrate its compatibility with a number of differentiation protocols, some of which can be seen on the screen now. We've successfully differentiated this line into cell types of all three embryonic germ layers, as well as 16 other cell types, including neurons, cardiomyocytes, and microglia, and organoids, including intestinal, cerebral, and brain region-specific organoids, and that was all performed using our stem diff kits. Finally, we also tested the 3A's ability to scale up in PBS bioreactors. Cells were expanded in teaser A-O-F3D, an animal origin-free bioreactor medium. The linear log cumulative fold expansion plot showed consistent expansion with no lag phase during transition into 3D, and we saw an average daily fold expansion of 1.5. In general, we find that a total of 1 billion viable cells is reached after about five passages. And with that, I'd like to say thank you very much for listening. SCTI-003A is available for use in your laboratory now, and if you'd like to explore further data and information about the line, feel free to use your phone camera to scan the QR code on your screen now or visit the website directly at stemcell.com forward slash SCTI-003-A. You can also contact us with any questions or feedback at IPSCRequest at stemcell.com. Thank you very much. | ↗ |