Prepare the media reagents:
1. Make up a 100 mg/ml stock of PVA in DI water in a heatproof glass bottle and autoclaving to dissolve (following autoclave machine instructions). Do not tighten glass bottle lid while autoclaving. After cooling, confirm PVA is completely-dissolved visually. Stock at 4°C.
2. Dissolve SCF and TPO cytokines at 1:1000 stocks in F12 media (10 ug/ml for SCF; 100 ug/ml for TPO). Aliquot and stock at -20°C or -80°C.
Make the HSC media:
1. Media should be prepared fresh for every use and media should be pre-warmed to 37°C before use.
2. Mix media reagents to make F12 media supplemented with 10mM HEPES, 1X P/S/G, 1X ITSX, 1 mg/ml PVA, 100 ng/ml TPO, and 10 ng/ml SCF. Prepare enough for 200 ul per well. Note that PVA solution is viscous and will need to be pipetted slowly. Mix media well by inversion before use.
3. Transfer 200 ul media into desired 96-well plate wells (or 1 ml media for 24-well plate wells). Fill remaining plate wells with PBS.
Initiate the HSC culture:
1. Directly sort HSCs by FACS into 96 well plate wells. For bulk HSC cultures, we recommend starting with at least 50 cells/well, although 1-500 cells can be sorted into a 96-well plate. Please see other published protocols for HSC sorting (6,7,8).
2. Although cultures can be initiated from mouse cKit+Sca1+Lineage-or CD34-/locKit+Sca1+Lineage-bone marrow cells, we recommend using CD150+CD34-/locKit+Sca1+Lineage-.
Maintain the HSC culture:
1. Following sorting, HSCs should be immediately transferred to a tissue culture incubator set to 37°C with 5% CO2 and 20% O2.
2. Avoid disturbing the cultures where possible, although cells can be visually evaluated by light microscopy.
3. After the first 5-6 days of culture, media changes should be made every 2-3 days. It is important to perform complete media changes on the cultures to avoid the buildup of differentiation-inducing cytokines.
4. Before initiating media changes, prepare fresh HSC media as above. Use a pipette to gently remove all media from the well. Remove media from the wall of the well and the media’s surface to avoid disturbing the cells, which are only gently attached to the well bottom. Remove all media until the meniscus reaches the plate bottom.
5. Gently add 200 ul of fresh and pre-warmed media down the well of the well. To avoid cells drying out, we do not recommend removing media from too many wells at one time.
6. After media changes, transfer back to the tissue culture incubator. Repeat media changes every 2-3 days throughout the culture.
7. Cultures initiated from 50 cells can be maintained in the same 96-well plate well for 28-days. Alternatively, once cells reach ~90% confluency, cells can be expanded. Typically, 1x 96-well plate well can be passaged into 3x 96-well plate wells (each containing 200 ul media) or 1 x 24-well plate well (containing 1 ml media). Cultures initiated from 50 cells typically reach ~90% confluency after 21 days of culture. Dissociated attached cells by gently pipetting the media over the plate bottom.
8. At any time during culture, cells can be used for in vitro analysis (e.g. cell counting, flow cytometric analysis, or other) or in vivo transplantation (see other published protocols on the HSC transplantation assay (6,7,8).
Cell counting analysis of the HSC culture:
1. Cell cultures can be counted at any point, although it is not recommended to repeatedly mix the HSC cultures.
2. Dissociate attached cells by gently pipetting the media over the plate bottom and then transfer 10 ul of the culture to a tube. Mix 1:1 with Trypan Blue and count using a hemocytometer.
3. Alternatively use an automated cell counter, following the manufacturer’s instructions.
4. After 1-week, 50 HSCs should generate approximately 1.5x104 cells in one 96-well plate well. After 28-days, 50 HSCs should generate approximately 4x105 cells in one 96-well plate well.
Flow cytometric analysis of the HSC culture:
1. Cell cultures can be assessed by flow cytometry by staining cells with Sca1, cKit, and Lineage antibodies. The concentration of each antibody should be individually titrated before use. Necessary antibody concentration will change between batches and vendors.
2. Dissociate attached cells by gently pipetting the media over the plate bottom (avoid generating bubbles). Transfer to a tube and spin down at 1500 rpm (440 g) for 5 minutes.
3. Resuspend cells in PBS containing a lineage antibody cocktail (containing Biotin-CD4, Biotin-CD8, Biotin-CD45RA/B220, Biotin-Ter119, Biotin-Ly6G/Ly6C, Biotin-CD127, Biotin-FceR1). Stain cells at 4°C for 30 minutes, wash with at least 10 volumes of PBS and spin down.
4. Resuspend cells in PBS containing PE-Sca1, APC-cKit, Streptavidin-APC/eFluor780 antibodies. Stain cell at 4°C for 30 minutes, wash with at least 10X volume of PBS, spin down.
5. Resuspend cells in ~200 ul PBS containing 1X PI, transfer to a FACS tube, and store at 4°C until analysis.
6. Analyze on a FACS machine/flow cytometer using unstained and single stained samples as controls, and quantify the frequency of Kit+Sca1+Lineage- live cell population.