Biomedical subjects
Publications and source records attributed to C A Heath.
Recent advances in stem-cell technology have improved the prognosis for tissue engineering. The use of cultured stem and/or progenitor cells has the potential to improve the extent of regeneration, and also increases the likelihood that the transplanted tissue will integrate with the surrounding tissue. It could eventually even reduce or eliminate the need for immunosuppressive drugs.
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Equine articular chondrocytes, embedded within a polyglycolic acid nonwoven mesh, were cultured with various combinations of intermittent pressure, fluid flow, and mixing to examine the effects of different physical stimuli on neochondrogenesis from young cells. The cell/polymer constructs were cultured first in 125 ml spinner flasks for 1, 2, or 4 weeks and then in a perfusion system with intermittent pressure for a total of up to 6 weeks. Additional constructs were either cultured for all 6 weeks in the spinner flasks or for 1 week in spinners followed by 5 weeks in the perfusion system without intermittent pressure. Tissue constructs cultivated for 2 or 4 weeks in spinner flasks followed by perfusion with intermittent pressure had significantly higher concentrations of both sulfated glycosaminoglycan and collagen than constructs cultured entirely in spinners or almost entirely in the pressure/perfusion system. Initial cultivation in the spinner flasks, with turbulent mixing, enhanced both cell attachment and early development of the extracellular matrix. Subsequent culture with perfusion and intermittent pressure appeared to accelerate matrix formation. While the correlation was much stronger in the pressurized constructs, the compressive modulus was directly proportional to the concentration of sulfated glycosaminoglycan in all physically stressed constructs. Constructs that were not stressed beyond the 1-week seeding period lost mechanical integrity upon harvest, suggesting that physical stimulation, particularly with intermittent pressure, of immature tissue constructs during their development may contribute to their ultimate biomechanical functionality.
Isolated equine chondrocytes, from juveniles and adults, were cultured in resorbable polyglycolic acid meshes for up to 5 weeks with semicontinuous feeding using a custom-made system to intermittently compress the regenerating tissue. Assays of the tissue constructs indicate that intermittent compression at 500 and 1000 psi (3.44 and 6.87 MPa, respectively) stimulated the production of extracellular matrix, enhancing the rate of de novo chondrogenesis. Constructs derived from juvenile cells contained concentrations of extracellular matrix components at levels more like that of native tissue than did constructs derived from adult cells. With intermittent pressurization, however, even adult cells were induced to increase the production of extracellular matrix. At both levels of intermittent pressure, the concentration of sulfated glycosaminoglycan in constructs from juvenile cells was found to be up to ten times greater than concentrations in control (nonpressurized) and adult cell-derived constructs. Whereas collagen concentrations in the 500 psi and control constructs were not significantly different for either juvenile or adult cell-derived constructs, intermittent pressurization at 1000 psi enhanced the production of collagen, suggesting that there may be a minimum level of pressure necessary to stimulate collagen formation.
A semi-continuous compression/perfusion system has been custom made to allow the application of intermittent hydrostatic pressure, at physiological levels, to regenerating tissues over the long term. To test the system, isolated foal chondrocytes were seeded in resorbable polyglycolic acid meshes and cultured in the system for 5 weeks. The cell/polymer constructs were subjected to an intermittent hydrostatic pressure of 500 psi and were fed semi-continuously. Assays of the resulting tissue constructs indicate that the reactor supports cartilage development and that physiological intermittent compression enhances the production of extracellular matrix by the chondrocytes. The concentrations of sulfated glycosaminoglycan were found to be at least twice as high as those in control (unpressurized) samples. A correlation between the sulfated glycosaminoglycan content and the compressive modulus in pressurized, but not control, samples suggests that physiological intermittent pressurization not only enhances the production of extracellular matrix but may also influence matrix organization resulting in a stronger construct.
This article describes recent, significant scientific advances leading to the development of the bioartificial nerve graft. Schwann cells, which play an active role in the repair and function of peripheral nerves, are used to seed a synthetic, often resorbable conduit, which is then used to bridge and repair nerve gaps caused by injury or disease. By enhancing the rate and extent of regeneration, the bioartificial nerve graft holds great promise for improving recovery in the peripheral (and central) nervous system.
Because our earlier work indicated a strong correlation between specific antibody productivity and cell density in perfusion culture, we conducted experiments to determine the optimum means of increasing cell density while maintaining high antibody productivity. The rates of medium supply and waste removal were varied to determine whether cell density was limited or inhibited, and whether a diffusable substance could be responsible for the correlation between antibody productivity and cell density. Nutrient supply was found to be a stronger determinant of cell density than waste removal; however, the rate of waste removal had a greater effect on cell growth at lower cell densities. Even at noninhibitory levels of ammonia and lactate, cellular metabolism was regulated to minimize their concentrations at lowered rates of waste removal. Separate step changes in glucose and glutamine resulted in increased cell density and antibody concentration. Specific antibody productivity increased following the step in glutamine, but not glucose. Both steps caused changes in cellular metabolism that prevented the levels of lactate and ammonia from reaching toxic levels.
A quadroma (#22 x 63), formed by the fusion of two hybridomas, and its parent hybridomas (#22 and FMC 63) were each grown in fed batch cultures in order to examine the change in antibody productivity over time of the quadroma compared to its parent hybridomas. The growth rate, glucose uptake rate and lactate production rate of the quadroma were found to be intermediate between those of its parent cells of origin. The specific antibody productivity and internal antibody content of the quadroma followed the same decreasing trends over time as those seen in both parent hybridomas. Losses in specific antibody production rate and antibody content, however, occurred at a faster rate for the quadroma than for either of its parent hybridomas. Although the growth of a non-producing subpopulation is presumed to account for the drop in antibody production, there was no direct correlation between the percentage of high antibody containing cells, as determined by flow cytometry, and the specific antibody production rate.
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Immunomagnetic beads can be used to remove subpopulations of cells from a mixed cell suspension in a flow-through system. One application of this process is the removal of tumor cells from bone marrow prior to its use in autologous bone marrow transplantation (ABMT). Based on preliminary data showing that three monoclonal antibodies (MoAb) (SCCL 175, HNK-1, and TFS-4) gave optimal separation in small-scale experiments, we have designed a large-scale separator suitable for clinical use. In our separator, the cell suspension flows through a 150 ml baffled transfer pack which is held over an array of permanent magnets. Direct (one MoAb only) and indirect (MoAb and anti-mouse antibody) methods of binding beads to cells were investigated as were the effects of temperature, bead to cell ratio, and medium additives on tumor cell removal and normal cell recovery. We determined the optimal separation conditions to be the indirect method of binding at 22 degrees C using a bead to tumor cell ratio of 25:1. Testing of the device on DMS-273 small cell lung cancer (SCCL) cells mixed with normal human bone marrow mononuclear cells resulted in a mean tumor cell removal of 3.64 logs (99.977%) with a concomitant mean normal granulocyte-monocyte colony forming unit (CFU-GM) recovery of 61.3%. These experiments form the basis to use the immunomagnetic beads to purify bone marrow from patients with SCCL for use in ABMT.
We have developed nuclear magnetic resonance (NMR) flow imaging techniques to measure fluid flow in a cell-free hollow fiber bioreactor (HFBR). Using 1H NMR we track the motion of protons and obtain velocity distributions as a function of position and time. These measurements enable the visualization of flow patterns needed for module design and for establishing desired operating conditions. Uneven flow in the cell-containing region of an HFBR can result in concentration gradients and uneven cell distribution that may lead to reduced cell viability. Results from this non-invasive method could be used to design more efficient cell bioreactors or membrane separation devices.
Antisheep antileukocyte serum (ALS) was produced in rabbits, purified, and adsorbed against erythrocytes and platelets. The ALS was infused intra-arterially over a 3-hour period into anesthetized sheep (n = 6) prepared with lung lymph fistulas. Pulmonary vascular resistance (PVR) and pulmonary lymph flow (Qlym) increased two- to threefold while the lymph-to-plasma protein concentration ratio (L/P) did not change from baseline, suggesting an increase in pulmonary vascular permeability to proteins. The arterial leukocyte count decreased from 4,935 +/- 840 to 1,385 +/- 325 cells/microliters, the neutrophil count decreased from 1,045 +/- 265 to 340 +/- 130 cells/microliters, and the platelet count decreased from 2.8 X 10(5) +/- 0.2 X 10(5) to 0.65 X 10(5) +/- 0.12 X 10(5) cells/microliters. ALS induced neutrophil aggregation in vitro, but not platelet aggregation. In the present study, we also examined the effects of ALS-induced leukopenia on the increase in pulmonary vascular permeability after intravenous alpha-thrombin challenge to assess the role of leukocytes in mediating the increased permeability response. Sheep (n = 5) were made leukopenic by an intramuscular injection of ALS; Qlym was in the normal range 4 to 5 hours after the ALS administration. In the leukopenic group, thrombin challenge caused an increase in Qlym from 7.8 +/- 0.7 to 12.9 +/- 1.9 ml/hr (a 64% increase) while L/P decreased from 0.86 +/- 0.04 to 0.70 +/- 0.05 (a 19% decrease). In contrast, thrombin-induced intravascular coagulation in control sheep (n = 5) produced a 250% increase in Qlym with an increase in L/P. The results indicate that leukocytes are required for the increase in lung vascular permeability after thrombin-induced intravascular coagulation.
Variable effects of cimetidine on the clearance in humans of the high-clearance compounds lidocaine and indocyanine green have been reported, some investigators finding a reduction and others no change. We measured the extraction of indocyanine green, which is not metabolized and of lidocaine, which is metabolized by the perfused rat liver, in an open system with a fixed flow rate. The extraction ratios of both indocyanine green (ERICG) and lidocaine (ERL) were determined under control conditions and during continuous infusion of cimetidine and other H2-receptor antagonists (ranitidine, nizatidine, and ICI 125,211) on separate occasions. The effects of increasing concentrations of cimetidine and ranitidine were measured, and single concentrations of nizatidine and ICI 125,211 were used. Indocyanine green was measured spectrophotometrically or by high-performance liquid chromatography (HPLC). Lidocaine concentrations in perfusate were measured by gas chromatography, and H2-receptor antagonist levels in perfusate and in liver by HPLC. The perfused rat liver extracted indocyanine green (ERICG = 0.43 +/- 0.04) and lidocaine (ERL = 0.78 +/- 0.01) with steady state being reached within 5 minutes. Neither cimetidine nor ranitidine altered steady-state indocyanine green extraction. In contrast, ERL was decreased by all four H2-receptor antagonists but with differing potencies. In this system, cimetidine was the most potent agent, reducing ERL by 28.5% at a cimetidine concentration of 56 mumol/L. The other H2-receptor antagonists also decreased ERL:ICI 125,211 by 20% (49 mumol/L), ranitidine by 13% (38 mumol/L), and nizatidine by 9% (43 mumol/L). A dose-response relationship for cimetidine and ranitidine was developed, confirming the greater potency of cimetidine.(ABSTRACT TRUNCATED AT 250 WORDS)
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Suspended mammalian cells can be cultivated in a variety of operational modes (pure chemostat, total cell retention, or partial cell retention) in a homogeneous perfusion bioreactor by varying the cell bleed rate. Hybridomas were grown in the reactor at a perfusion rate of 2.0 day-1 for over 10 weeks at different specific growth rates and viable cell densities achieved by varying the extent of cell retention. Cell metabolism in the reactor was found to vary with the extent of cell retention, which determined both cell density and specific growth rate. With partial cell retention, the nutrient consumption and metabolite production rates decreased with both increasing growth rate and increasing cell density. The specific and volumetric antibody production rates, however, increased dramatically with cell density (and to a lesser extent with decreasing growth rate). The specific MAb production rate was lower with total cell retention than with partial retention at the same growth rate. Since the reactor can be operated over a range of perfusion rates and extents of cell retention, the system can be used to culture cell lines with widely different productivity patterns.