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. Mar-Apr 2011;27(2):451-9.
doi: 10.1002/btpr.551. Epub 2011 Feb 2.

Use of a Centrifugal Bioreactor for Cartilaginous Tissue Formation From Isolated Chondrocytes

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Free PMC article

Use of a Centrifugal Bioreactor for Cartilaginous Tissue Formation From Isolated Chondrocytes

Christopher J Detzel et al. Biotechnol Prog. .
Free PMC article

Abstract

Although a centrifugal bioreactor (CCBR) supports high-density mammalian suspension cell cultures by balancing drag, buoyancy, and centrifugal forces, to date anchorage-dependent cultures have not been tried. Also, steady or intermittent hydrostatic pressures of 8 to 500 kPa, and shears of 0.02 to 1.4 N/m(2) can be simultaneously applied in the CCBR. This article demonstrates the use of a CCBR to stimulate chondrogenesis in a high-density culture. At 3 weeks, histological results show even distribution of glycosaminoglycan (GAG) and collagen, with 1,890 ± 270 cells/mm(2) cell densities that exceed those of 1,470 ± 270 in pellet cultures. Analysis of collagen content reveals similar levels for all treatment groups; 6.8 ± 3.5 and 5.0 ± 0.4 μg collagen/μg DNA for 0.07 and 0.26 MPa CCBR cultures, respectively, in contrast to 6.6 ± 1.9 values for control pellet cultures. GAG levels of 5.6 ± 1.5 and 4.1 ± 0.9 μg GAG /μg DNA are present for cultures stressed at 0.07 and 0.26 MPa, respectively, in comparison to control pellet cultures at the 8.4 ± 0.9 level. Although results to date have not revealed mechanical stress combinations that stimulate chondrogenesis over unstressed controls, system advantages include continuous culture at cell densities above those in the pellet, precise medium control, the ability to independently vary multiple mechanical stresses over a broad range, and the flexibility for integration of scaffold features for future chondrogenesis stimulation studies.

Figures

Figure 1
Figure 1. Cell purging tubing sets were modified to reflect the configuration shown in the schematic of the CCBR and fluid lines
Fresh medium was supplied to the reactor chamber during centrifugation with the use of flexible tubing and an anti-twister device. During the 3-week culture, the waste pump was set to remove 200 mL/day, which was replaced with fresh culture medium from the fresh feed (FF) tank.
Figure 2
Figure 2. After 3 weeks of culture in the CCBR, a cartilage construct has formed from the original 2.5 × 108 isolated chondrocytes seeded into the bioreactor
The construct is observed before (A) and after (B) removal from the reactor chamber. The reactor chamber containing the tissue construct (A) is viewed from above with direction of rotation to the left; with reference to this photo the axis of rotation is perpendicular to the plane of the page.
Figure 3
Figure 3. Histological results show cells are round and evenly distributed throughout the ECM of both the pellet and CCBR culture constructs, although the cell density is higher in the CCBR construct
Toluidine blue stain for GAG content is shown in (A) and an increase in staining intensity is difficult to discern in the pellet culture when comparing Week 1 and Week 3 sections, but an increase in intensity is clearly seen in sections of the CCBR construct. A trichrome stain (B) for collagen shows collagen incorporation is increased into the ECM of both constructs over time. (Bar represents 50 μm).
Figure 4
Figure 4. Collagen content normalized to DNA shows significant increases in collagen content from Week 1 to Week 3 for both the pellet and bioreactor culture systems
At Week 1, pellet cultures contain 2.8 ± 0.6 μg/μg collagen/DNA while bioreactor cultures contain 1.8 ± 0.4 μg/μg collagen/DNA at the same time point. These values increase to 6.6 ± 1.9 μg/μg for pellet cultures at 3 weeks while bioreactor cultures increase to 6.8 ± 3.5 and 5.1 ± 0.4 μg/μg for the 900 and 1650 rpm procedures, respectively. (* indicates P < 0.05)
Figure 5
Figure 5. Biochemical results of GAG content are normalized to DNA content for comparison of CCBR cultures as cell density varies between culture systems
GAG/DNA content is significantly higher in pellet cultures (n = 7) at the 1-week time point, with a value of 7.0 ± 1.1 μg/μg, when compared to the value for CCBR constructs (n = 2), 1.4 ± 0.1 μg/μg. The GAG/DNA content increases from Week 1 to Week 3 in all treatment groups, with pellet cultures containing, 8.4 ± 0.9 μg/μg GAG/DNA, and CCBR constructs containing; 5.6 ± 1.5 μg/μg at 900 rpm (n = 2) and 4.1 ± 0.9 μg/μg for the 1650 rpm (n = 2) culture condition. (* indicates P < 0.05)

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