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Human Umbilical Cord Blood Stem Cells are positively selected from umbilical cord blood (CB) mononuclear cells (MNCs). Whole umbilical cord blood is needle aspirated from the umbilical cord vein using a cord blood collection bag containing 35 mL of CP2D the anticoagulant citrate phosphate double dextrose. Mononuclear cells are enriched from the cord blood using a density gradient centrifugation protocol. CD133+ HSCs) hematopoietic stem cells( are then selected using immunomagnetic anti-CD133 microbeads from the mononuclear cell pool, leaving a highly pure population of CD133+ stem and progenitor cells. At Stemell we offer single and mixed donor stem cells upon request. Mixed donor pools are from two or more donors and are available in larger lots. Single donor cells are obtained from a single umbilical cord sample. Cells were obtained using IRB (Institutional Review Board) approved consent forms and protocols.

LiveCyte products allow for prolonged storage before use. Cell products contain 10% DMSO to minimize cell death during freezing. All cryopreserved products are stored in containers designed and tested for ultra-low temperatures at long time intervals. We normally ship LiveCyte products on dry ice.

All samples are collected at nearby partner hospitals or clinics. Samples are then quickly processed in our on-site laboratory in the city of San Juan Capistrano to achieve maximum viability and quality. Stemell cells are frozen at -1°C/minute in a -80°C freezer, and then transferred to liquid nitrogen.

Newcyte cells should be maintained in liquid nitrogen at -135°C. The cells are warrantied for 6 months from the date of receipt if stored at -135°C or colder. Storage of cells at -80°C for less than one month should maintain cell viability but is not covered by the warranty.

• Frozen Newcyte Vial containing cells
• Desired medium warmed to 37°C
• Sterile 50 mL centrifuge tube
• Water bath warmed to 37°C
• 70% ethanol
• DNase I

1. Place the vial into the 37°C water bath. You may consider leaving the vial in a sealed plastic bag to reduce the chance of contamination. Perform this step immediately after removing the vial from the dry ice in the shipment or after removing the vial from your liquid nitrogen storage.
2. Quickly thaw the cells in less than a minute by gently swirling the vial in the water bath until only a small bit of ice remains. Do NOT vortex the cells at any point while thawing, and work quickly to maximize cell viability.
3. Wipe the vial with 70% ethanol and transfer it to a laminar flow hood.
4. Gently mix the cells by inverting the vial. Measure the cell suspension volume in the vial.
5. Aliquot 10 μL of cell suspension from the vial using aseptic technique; have a separate person proceed with the cell count and viability measurement using the counting Instructions provided. Important: This cell viability/counting step is required to ensure the quantity of cells provided. Thawing and counting must be completed simultaneously with two people to ensure accurate measurements in the cell count and to maintain product viability. Be sure to count the cells at this step, before washing, because you will lose cells in the wash.
6. Transfer the cell suspension to the sterile centrifuge tube containing DNase I, which will prevent cell clumping. For mononuclear cells, use cell suspension. DNase I is not necessary if cells are to be lysed for protein or DNA/RNA extraction.
7. Rinse the remaining cells from the vial with 1 mL warmed medium. Slowly add this suspension to the centrifuge tube with the cells in a dropwise fashion, 1 drop every 5 seconds, while gently swirling the tube.
8. Add 15–20 mL warmed medium to the cells. Gently mix by inverting the closed tube.
9. Wash the cells by centrifuging at room temperature, 300 rcf for 10 minutes, with low brake.
10.Check the supernatant for clarity, and check the bottom of the tube for a complete cell pellet. Carefully remove the supernatant with a pipette, leaving a small volume so as to not disturb the pellet. Resuspend the
11. Again add 15–20 mL warmed medium and perform the wash step once more.
12.Gently resuspend the cells in warmed medium. They are now ready for use.
Important: Be aware that cell loss is expected and may be up to 30% during thaw and wash steps. Recovery rates vary depending on technique.

Important: This cell viability/counting step is required to ensure the quantity of cells provided. Be sure to count the cells before washing. Be aware that cell loss is expected and may be up to 30% during wash steps. Recovery rates vary depending on technique.
• Cleaned hemocytometer
• Trypan Blue
1. If removing the cell suspension from the vial in which it was shipped, be sure to rinse the vial to collect all of the cells.
2. Gently mix the cell suspension and measure the volume.
3. Make a 1-in-2 dilution with 20 μL each of well-mixed cell suspension
4. Load one side of the hemocytometer, being careful not to over- or under-fill the chamber.
5. Count viable (clear, round, bright) and non-viable (blue, irregular shape, dull) cells in the four corner squares. Adjust your dilution if there are more than 100 cells/square.
6. Determine the number of total viable cells in the original sample. One square is equal to 100 nL. Viability = live cells/all cells Cell Concentration = Mean cells/square × Dilution Factor × 104 Total Cell Count = Cell Concentration × Starting Volume

This product contains human tissue or other biological material and MUST be handled at Biosafety Level 2 or higher. All biological products should be treated as potentially infectious or contaminated material, even if infectious disease screening reports are negative. Follow universal precautions and wear appropriate personal protective equipment.

Newcyte warranties its fresh products if tested immediately upon receipt and if counted exactly as in the above instructions. The cells are provided the above instructions are followed exactly. Newcyte is not able to guarantee cell performance for any in vitro or in vivo culture system, proliferation assay, functional assay, or implantation.