Abstract

The recipient of expanded hematopoietic cells, especially cord cells, could benefit from both self-renewing and differentiated cells, to enhance the speed and durability of engraftment. For this and other clinical and laboratory purposes, we developed a modular perfusion bioreactor system using a 3D scaffold for cell growth. The polycarbonate bioreactor body design allows media to flow over and under but not through disks of nonwoven polyethylene terephthalate (PET) fabric scaffold, and allows nonadherent progeny to fall from the matrix into the flow path. Gas-permeable membranes over and under the media path allow gas exchange. We developed several media supply and cell collection methods, including recirculating (R) and single pass (SP) modes. A modified recirculating mode (MR), in which the external gas exchanger and cell settler used in the recirculating mode were eliminated, also was tested, with or without a fluid reservoir in the flow loop. We tested the system using light density marrow cells or cd34 positively selected marrow or cord cells seeded directly or on preestablished marrow stromal cells (MSCs) or the HS-5 cell line, during runs lasting over 60 days. We analyzed nonadherent cells and cell-matrix constructs using flow cytometry; bright field, phase, confocal, and scanning electron microscopy of cytospins, the constructs themselves, and constructs embedded in wax and sectioned, using conventional and immunofluorescent staining; and clonogenic assays for committed hematopoietic cells. Harvested nonadherent cell output increased with experience with the developing bioreactor system, reaching up to 100x the number put into the device, with CD34+ % ranging from 0.3 to 14.3.

Selected experimental results, in chronologic order. All counts are per bioreactor module.

ExptMethodNonfeeder Cells In x106Total Nonadherent Cells Out x106 over (days)Viability (%)CD34+ %CD34+ lineage-%
Marrow CD34+ on MSC, R 0.9 8.7(68) 3.8 to 96 0.8 to 1  
Marrow CD34+ on MSC, SP 0.9 1(54) 38 to 93 0.24 0.2 
Same,R 0.9 7.1(55) 70 to 98 0.5–7.4 0.1 to 1.7 
Marrow CD34+ on MSC, R 0.5 68(68) 55 to 99 0.4–3.4 0.2 to 2.4 
Marrow CD34+ on MSC, MR 0.5 13(68) 14 to 94 0.4 to 3.2 .3 to 1.4 
Marrow light density, MR+ 23 51(38) 78 to 100 1.6 to 2.6 .3 to 2.3 
Cord CD34 + on MSC, MR 0.9 21(60) 68 to 95 0.3 to 14.3  
Cord CD34 + on HS-5, MR 0.9 30(59) 4 to 68   
ExptMethodNonfeeder Cells In x106Total Nonadherent Cells Out x106 over (days)Viability (%)CD34+ %CD34+ lineage-%
Marrow CD34+ on MSC, R 0.9 8.7(68) 3.8 to 96 0.8 to 1  
Marrow CD34+ on MSC, SP 0.9 1(54) 38 to 93 0.24 0.2 
Same,R 0.9 7.1(55) 70 to 98 0.5–7.4 0.1 to 1.7 
Marrow CD34+ on MSC, R 0.5 68(68) 55 to 99 0.4–3.4 0.2 to 2.4 
Marrow CD34+ on MSC, MR 0.5 13(68) 14 to 94 0.4 to 3.2 .3 to 1.4 
Marrow light density, MR+ 23 51(38) 78 to 100 1.6 to 2.6 .3 to 2.3 
Cord CD34 + on MSC, MR 0.9 21(60) 68 to 95 0.3 to 14.3  
Cord CD34 + on HS-5, MR 0.9 30(59) 4 to 68   

Seventy to 100 % of cells collected were CD45+, CD14+ 20–48%, CD41 0.5–9%, CD19+ 0–2.6%, and CD3+ 0–1.6%. Confocal and SEM studies of the cell-matrix constructs demonstrated extracellular matrix deposits containing collagen, predominately types I and II, and fibronectin. This was most concentrated over the surfaces of the cell-matrix constructs, possible due to oxygenation gradients. CFU-GM were identified in the output for up to 65 days. This modular 3D perfusion bioreactor allows continued hematopoiesis over 60 days, with evidence of maintenance of early progenitors (by continued CFU-GM and CD34+ cell output) during that time. It may be an ideal device for studies of normal and perturbed hematopoiesis and for clinical use.

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