Collection of hematopoietic CD34 stem cells in rhesus macaques using Spectra Optia

Abstract

Background Non-human primates, particularly rhesus macaques, are ideal pre-clinical large animal models to investigate organ tolerance induction protocols using donor hematopoietic stem cells (HSCs) to induce microchimerism. Their relatively small size poses some challenges for the safe and effective collection of peripheral blood HSCs through apheresis procedures. We describe our experiences using the Spectra Optia apheresis unit to successfully obtain HSCs from mobilized peripheral blood of rhesus macaques. Method Mobilization of peripheral blood HSCs was induced using granulocyte stimulating factor (G-CSF) and Mozobil. The Spectra Optia unit was used in 18 apheresis procedures in 13 animals (4.9–10 kg). Animal health was carefully monitored during and after the procedure. Changes in peripheral blood cells before, during and after procedure were determined by complete blood count and flow cytometry. Results The automatic settings of the Spectra Optia unit were applied successfully to the procedures on the rhesus macaque. All animals tolerated the procedure well with no mortality. Mobilization of HSCs were most consistently achieved using 50μg/kg of G-CSF for 5 days and a single dose of Mozobil on the 5th day, followed by collection of cells 3 hours after Mozobil injection. The final apheresis product contained an average of 23 billion total nucleated cells with 47% granulocytes, 3,871 million total CD3 cells and 77 million CD34 cells which resulted in an average of 10 million CD34+ cells/kg of donor weight. Conclusion Apheresis of peripheral blood mobilized HSCs in rhesus macaques using Spectra Optia is a safe and effective procedure.

Safety and efficacy of autologous mononuclear cell and stem cell apheresis in very low‐weight children—Experience at a single center

“…The median CD34+ CE2 with the Optia in a series of 243 reported procedures was 48%, but most donors were adults, and none of the patients required the blood priming procedure . As observed in animal experiments, the results of performing blood‐primed MNC procedures in macaques with a weight of 4 to 10 kg resulted in a median CD34+ CE2 of 37% (range 13%‐67%), which was lower than that in adults and similar to that in our patients . The CE2 predicts the final CD34+ cell yield basing on assumption of stable cell counts during apheresis, and increased number of processed blood volumes in low body weight subjects can result in decline of circulating CD34+ cell numbers, that are not replenished by the in‐procedure mobilization, leading to lower efficacy of cell collections.…”

Comparison of three mobilization protocols for peripheral blood stem cell apheresis with Spectra Optia continuous mononuclear cell protocol in healthy dogs

“…In the present study, peripheral PLT counts and haematocrit decreased after plerixafor administration in some dogs. In the rhesus macaque model study, subcutaneous injection of plerixafor resulted in a decrease in peripheral PLT counts . Furthermore, plerixafor influences red blood cells and PLTs; it reportedly causes anaemia or thrombocytopenia in 3% to 6% of human patients .…”

“…Variations in apheresis parameters and/or the timing of the collection of cells may have contributed to the failure to obtain a sufficient number of PBSCs in our study. In a similar study using a rhesus macaque model, the apheresis procedures were initiated 3 to 6 hours after plerixafor administration . In the canine transplantation model study, the number of circulating PBSCs peaked at 8 hours following plerixafor administration in three of four dogs .…”

“…In general, the peripheral blood of various animals, such as humans, rhesus macaques and dogs, contains a small number of HSCs . In both human and veterinary medicine, granulocyte colony‐stimulating factor (G‐CSF) is the most commonly used agent for facilitating PBSC mobilization .…”

Terumo spectra optia leukapheresis of cynomolgus macaques for hematopoietic stem cell and T cell collection

“…G-CSF dosing was increased from 10 to 50 μg/kg G-CSF because a recent report demonstrated higher blood CD34+ cell counts in rhesus macaques mobilized with 50 μg/kg G-CSF dosing compared to 10 μg/kg or 100 μg/kg. 8 Daily small blood draws were taken for complete blood count (CBC) and flow cytometry staining to determine frequencies and absolute numbers of CD34+ hematopoietic stem cell progenitors. Briefly, EDTA-treated whole blood (50-100 μL) was washed twice with PBS and stained for CD45 (PE-Cy7, clone D058-1283, BD, Franklin Lakes, NJ), CD3 (AF700, clone SP34-2, BD), CD34 (PE, clone 561, Biolegend, San Diego, CA), CD45RA (V450, clone 5H9, BD), CD90 (FITC, clone 5E10, Biolegend), and viability (Live/dead Yellow Fixable, Invitrogen, Carlsbad, CA) at room temperature for 30 minutes.…”