Compartmental hollow fiber capillary membrane-based bioreactor technology for in vitro studies on red blood cell lineage direction of hematopoietic stem cells

Abstract

Continuous production of red blood cells (RBCs) in an automated closed culture system using hematopoietic stem cell (HSC) progenitor cell populations is of interest for clinical application because of the high demand for blood transfusions. Previously, we introduced a four-compartment bioreactor that consisted of two bundles of hollow fiber microfiltration membranes for transport of culture medium (forming two medium compartments), interwoven with one bundle of hollow fiber membranes for transport of oxygen (O(2)), carbon dioxide (CO(2)), and other gases (forming one gas compartment). Small-scale prototypes were developed of the three-dimensional (3D) perfusion cell culture systems, which enable convection-based mass transfer and integral oxygenation in the cell compartment. CD34(+) HSC were isolated from human cord blood units using a magnetic separation procedure. Cells were inoculated into 2- or 8-mL scaled-down versions of the previously designed 800-mL cell compartment devices and perfused with erythrocyte proliferation and differentiation medium. First, using the small-scale 2-mL analytical scale bioreactor, with an initial seeding density of 800,000 cells/mL, we demonstrated approximately 100-fold cell expansion and differentiation after 7 days of culture. An 8-mL laboratory-scale bioreactor was then used to show pseudocontinuous production by intermediately harvesting cells. Subsequently, we were able to use a model to demonstrate semicontinuous production with up to 14,288-fold expansion using seeding densities of 800,000 cells/mL. The down-scaled culture technology allows for expansion of CD34(+) cells and stimulating these progenitors towards RBC lineage, expressing approximately 40% CD235(+) and enucleation. The 3D perfusion technology provides an innovative tool for studies on RBC production, which is scalable.

FIG. 1.

Internal structure of hollow-fiber bioreactor. (A) Shown is the arrangement of capillary layers in the bioreactor and a closer look at the smallest capillary unit with medium capillaries that are counter-currently perfused (red and blue) with integrated gas capillaries (yellow). (B) Scale-down of the technology from clinical scale (800 mL) to laboratory scales (8 or 2 mL) by varying the number and length of capillary layers. Color images available online at www.liebertonline.com/tec

FIG. 2.

Bioreactor and perfusion unit prototypes. Bioreactor perfusion system with pump heads for medium recirculation (left) and medium substitution (right); a heating chamber for bioreactor maintenance; rotameters for air, oxygen, CO₂, and total gas flow; and a display for digital monitoring and regulation of perfusion parameters. Bioreactor prototypes with different cell compartment volumes (from the left to the right: 800, 8 and 2 mL) are shown in the front. Color images available online at www.liebertonline.com/tec

FIG. 3.

Bioreactor perfusion schematic. A detailed schematic of the perfusion periphery connected to the laboratory-scale bioreactor. The bioreactor was integrated into a processor-controlled perfusion device with electronic flow/pressure-controlled perfusion, media, and waste pump operation. A heating unit provided a constant temperature of 37°C within the perfusion circuit. Flow rates of air, O₂, CO₂, and N₂ were controlled by a gas-mixing unit on the perfusion device. Color images available online at www.liebertonline.com/tec

FIG. 4.

Pseudo-continuous production study time course. CD34⁺ HSCs were cultured for 9 days in 2D Petri dish cultures and then 1.5×10⁷ total cells (1.8 million cells/mL) were inoculated into an 8-mL 3D bioreactor culture. Culture maintenance was performed according to lactate production, glucose consumption, and LDH levels as previously described. Intermediate cell harvests were performed on days 5, 7, 9, 11, and 13. After each harvest, the original density of cells was reinoculated into the bioreactor. HSCs, hematopoietic stem cells; 2D, two-dimensional; 3D, three-dimensional; LDH, lactate dehydrogenase. Color images available online at www.liebertonline.com/tec

FIG. 5.

(A) Schematic for semi-continuous 8-mL bioreactor study. After 2 days in 2D Petri dish culture cells were inoculated into an 8-mL bioreactor (BR001) at a density of 800,000 cells/mL and cultured for 5 days until lactate production reached a maximum level. Cells harvested from BR001 were passaged into a new bioreactor (BR002) at a density of 800,000 cells/mL with a separate fraction of the cells being reinoculated into BR001 at the original density. The production process was carried out for another passage, splitting cells into BR003, before final harvest on day 19. (B) Lactate production levels for BR001 during the semi-continuous production study. Points at which the reactor was passaged and harvested are indicated along with the corresponding lactate levels. The reactor was passaged according to observed peaks in lactate production and harvested when the lactate level dropped, according to the culture management strategy outlined in the Materials and Methods section. Color images available online at www.liebertonline.com/tec

FIG. 6.

Control cultures in 2D. Fold expansion of 2D Petri-dish control cultures maintained at a density of 20,000 cells/mL was used to check for infection and proper cell growth. Results are presented as a mean of n = 5 cultures ± standard deviation.

FIG. 7.

Glucose consumption and lactate production trends. Glucose consumption (mg/dL) and lactate production (mg/dL) for high-density 2-mL-scale bioreactors (n = 3) inoculated at a density of 800,000 cells/mL and cultured for 7 days after 2 days of Petri dish preculture. Cultures were harvested when lactate production and glucose consumption levels reached a peak and started to plateau. In addition to the measurements of glucose and lactate, the culture pH was monitored daily and maintained between 7.35 and 7.45 by adjusting the air/CO₂ gas ratio while maintaining the same overall gas flow rate.

FIG. 8.

Wright-Giemsa staining images. Cell samples were stained before inoculation (day 0) and after recovery (day 7) for a bioreactor from the high-density 2-mL bioreactor study. CD34-positive hematopoietic stems cells are shown at day 0, while late progenitor hematopoietic cells can be seen at day 7. Color images available online at www.liebertonline.com/tec

FIG. 9.

Cell surface antigen profiles from FACS and viabilities for intermediate harvests from the pseudo-continuous production study. After each intermediate cell harvest (days 5, 7, 9, and 11) FACS analysis was performed for CD34-FITC, CD71-APC, CD235a-PE, and CD45-PE markers. The inoculated cells at day 0 were pre-cultured over 2 days in 2D Petri dishes, which explains why the CD34+ cells were already differentiated. Cells rapidly lost CD34 expression and expressed high levels of CD71 (proliferative marker) while showing some expression of a characteristic RBC marker (CD235a). FACS, fluorescence-activated cell sorting. Color images available online at www.liebertonline.com/tec

FIG. 10.

Cell surface antigen profiles from FACS for final cell harvest from the pseudo-continuous production study. On the final day of harvest (day 13), 30 mL of medium was aspirated from the cell compartment three consecutive times (fractions 1, 2, and 3) and then finally 400 mL of fresh IMDM medium (fraction 4) was rinsed through the cell compartment. During each fractional harvest, cell samples were collected and FACS analysis was performed for CD34-FITC, CD71-APC, CD235a-PE, CD45-PE, and CD235a plus enucleation markers. Surface marker expression was similar among all fractions and gave a minimum of 30% mature RBCs (CD235a+ enculeation). The complete contents with the bioreactor cell compartment were removed using a fractional harvest method (orange x–y scatter line). Color images available online at www.liebertonline.com/tec

FIG. 11.

Cells harvested from an 8-mL bioreactor after 16 days in culture were analyzed using immunofluoroesence, scanning electron microscopy (SEM), and FACS. For immunofluorescence the following colors were used for staining: Phalloidin (green) for the presence of actin, CD235a (red) for the characteristic marker present on RBCs, and DAPI for the presence of a nucleus. We were able to capture a nucleus being expelled from a cell (white arrow). Interestingly, the SEM image below also shows a cell that is invaginating in preparation for ejection of the nucleus. FACS results indicate that these cells have lost the CD34⁺ marker, are in a proliferative stage of growth, and possess markers (CD235a and enucleation) characteristic of mature RBCs. The above image was adapted from Benjamin Cummings, an imprint of Addison Wesley Longman, Inc. Color images available online at www.liebertonline.com/tec