High efficiency microfluidic purification of stem cells to improve transplants
a technology of stem cells and microfluidics, applied in the field of high efficiency microfluidic purification of stem cells to improve transplants, can solve the problems of limiting the treatable patient population, reducing the success rate of engraftment, and increasing the risk of harmful side effects, so as to improve the quality of ucb and other transplant grafts, improve patient outcomes, and improve the effect of quality
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example 1
Leukocyte Enrichment from UCB
[0168]The methods can improve stem cell banking and transplantation by providing an efficient and robust processing system for clinical UCB, PBSC and BM harvests. The microfluidic separation method can efficiently and consistently deplete erythrocytes from UCB. In some cases, there may be problems with cell clumping in some clinical samples (principally due to dead / dying cells). In such cases, the device and / or protocol are optimized to address cell clumping. In some embodiments, the process is scaled up to purify >100 ml volumes of UCB per hour, preserving 90 / 90 / 90 performance.
[0169]In some cases, the blood sample is depleted of smaller-sized cells (i.e. erythrocytes, platelets) and the larger-sized cells of interest (i.e. leukocytes) are concentrated. Note that the unwanted smaller cells are present in blood at >1000-fold excess numbers over the desired leukocytes.
[0170]The microfluidic chips used can be approximately the size of a microscope slide. Th...
example 2
Characterize Performance of the Microfluidic Cell Separation Device with UCB
[0174]Anticoagulated, deidentified UCB samples are obtained. Samples with visible macroscopic cell clumps are classified as inadequate and not processed further; the numbers of inadequate samples are tracked. For adequate samples, UCB samples are diluted in an equal volume of running buffer and filtered through a 20 micron strainer before microfluidic processing. Recovered output (vs filtered input) cells are rigorously analyzed. Erythrocytes, leukocytes, and leukocyte subsets are quantified by Coulter and Hemavet technologies. Viability of output leukocytes are confirmed by trypan blue dye exclusion with counting by manual and automated (Countess) methods. Apoptosis and cell death are measured using Annexin V / 7AAD staining and flow cytometry. Leukocyte subtypes are quantified by immunostaining and flow cytometry. The number of CD34+HSPCs are evaluated using Procount kits.
[0175]Optical imaging tools (FIG. 17...
example 3
Increase Throughput to >100 ml / hr
[0178]In some cases, the throughput rate is scaled from 10 ml / hr in the system to >100 ml / hr. The most straightforward approach is to run the chips at a higher pressure differential. The system can operate at ˜5 mm / sec fluid speed in the chips. Increasing the driving pressure, the DLD method works well at speeds of at least 150 mm / sec (a 30× increase) to separate leukocytes from adult blood, while still maintaining 99% viability of the leukocytes. This speed corresponds to a chip throughput of 300 ml / hour. (And human cancer cells (mdamb231 cell line) have been processed at speeds up to 1000 mm / sec, also still maintaining 99% viability).
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