Growth of Canine Bone Marrow and Peripheral Blood Mononuclear Cells in In Vivo Plasma Clot Diffusion Chambers in Mice: Cytopoietic Activity, GM-CFC Content, and Physical Separation
Normal and regenerating canine bone marrow (BM) and normal peripheral blood mononuclear cells (PBMC) were cultured in vivo liquid (LDC) and in plasma clot diffusion chambers (PCDC) implanted intraperitoneally within sublethally irradiated mice. The cytopoietic activities (CA) (ratio of nucleated cells harvested per DC at day 7 of culture to the nucleated cell input) of BM and PBMC were significantly greater when grown within the plasma clot rather than liquid diffusion chamber cultures. BM and PBMC grown in plasma clot cultures had respective CAs of 2.8 and 1.3, while BM and PBMC grown in liquid cultures each had a respective CA of 1.2 and 0.7. Linear relationships were observed between the number of nucleated cells inoculated and the number of cells harvested after 6–8 days of culture.
Morphologically, both types of cultures resulted in predominant growth of granulocytes, monocytes, and macrophages. The cellular growth curves of BM in LDC and PCDC cultures were markedly different. Total nucleated cells per DC in liquid cultures decreased after implantation to a minimum value at day 4 and then rose to control levels by days 7–8. In contrast, the cellularity of PCDC cultures increased within 24 h and continued to rise, reaching plateau levels (two- to fourfold input) at days 6–8 of culture.
GM-CFC measured by in vitro agar culture could not be detected in suspensions derived from liquid DC cultures, whereas GM-CFC derived from PCDC cell suspensions remained at concentrations equivalent to input levels over 48 h and then decreased slowly through day 7 of culture.
Separation of canine density-“cut” BMC by velocity sedimentation indicated that the population of cells responsible for growth within PCDC is heterogeneous and that a subpopulation of these cells could be separated from GM-CFC. This PCDC population sedimented more slowly (3.7 mm/h)than the GM-CFC (5.5 mm/h). A second and more rapidly sedimenting peak was also observed for cells with CA and PCDC at 6.4 mm/h. There was significant overlap with the range of GM-CFC.
The data showed a significant advantage in growth of canine BMC and PBMC in plasma clot DCs over liquid DCs in terms of cellularity and growth of GM-CFC. Velocity sedimentation results indicated a separation of a population of cells responsible for growth in PCDC from the in vitro GM-CFC population. This may implicate a parent-progeny relationship between these cells similar to that recently described with human BMC in fibrin clot DCs (12).
Key Wordsgranulopoiesis diffusion chambers plasma clot canine
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