Hemoglobin regulates the metabolic and synthetic function of rat insulinoma cells cultured in a hollow fiber bioreactor.

Pancreatic islet transplantation continues to benefit patients with type 1 diabetes by normalizing glucose metabolism and improving other complications of diabetes. However, islet transplantation therapy is limited by the inadequate availability of pancreatic islets. In order to address this concern, this work investigated the expansion of rat insulinoma cells (INS-1) and their ability to generate insulin in a hollow fiber bioreactor (HFB). The long-term goal of this project is to develop a bioartificial pancreas. HFBs were incubated at two different oxygenation conditions (10% and 19% O(2)) to determine the best scenario for O(2) transport to cultured cells. Also, bovine hemoglobin (BvHb) was supplemented in the cell culture media of the HFBs in order to increase O(2) transport under both oxygenation conditions. Our results show that INS-1 cells expanded under all oxygenation conditions after 2 weeks of culture, with a slightly higher cell expansion under normoxic oxygenation (19% O(2)) for both control HFBs and BvHb HFBs. In addition, cellular insulin production remained steady throughout the study for normoxic control HFBs and BvHb HFBs, while it increased under hypoxic oxygenation (10% O(2)) for both types of HFBs but to different extents. Under the two different oxygenation conditions, cellular insulin production was more uniform with time in BvHb HFBs versus control HFBs. These results, along with qRT-PCR analysis, suggest a possible dysregulation of the insulin-signaling pathway under hypoxic culture conditions. In conclusion, the HFB culture system is an environment capable of expanding insulinomas while maintaining their viability and insulin production capabilities.

Immune recognition of exposed xenoantigens on the surface of PEGylated bovine red blood cells.

Due to potential problems that can occur during blood transfusion and increasing blood shortages, our group engineered methoxypolyethylene glycol conjugated bovine red blood cells (mPEG-bRBCs) as a potential universal oxygen therapeutic. This current work investigates the immunological properties of mPEG-bRBCs incubated with human plasma (hP) and correlates these properties to exposed Galalpha(1,3)Gal xenoantigens. After mPEG-bRBCs were incubated with hP, the amount of bound IgG and IgM was assessed via flow cytometry. Flow cytometry also assessed the amount of GS-IB4 bound to exposed Galalpha(1,3)Gal xenoantigens. The results of this study demonstrate that most hP samples strongly promote agglutination of mPEG-bRBCs regardless of the extent of mPEG surface coverage or donor blood type. IgG and IgM from hP bound strongly to mPEG-bRBCs. In general, the Galalpha(1,3)Gal xenoantigen remains exposed at all levels of PEG surface coverage. PEGylation did block some of the xenoantigens as the amount of exposed Galalpha (1,3)Gal decreased with increased mPEG surface coverage. However, this was not sufficient to prevent a strong agglutination reaction. Taken together, the results of this study indicate that the current strategy for PEGylating bRBCs is unsatisfactory for the development of immunologically silent oxygen therapeutics.

Hemoglobin-based oxygen carrier enhanced tumor oxygenation: a novel strategy for cancer therapy.

A tumor's low oxygen tension is a major obstacle to current chemo- and radio-therapies. By increasing oxygen transport to hypoxic tumor regions it is possible to make tumors more susceptible to current cancer treatment strategies. This article theoretically investigates the possibility of supplementing human blood with hemoglobin-based oxygen carriers (HBOCs) in an attempt to target oxygen delivery specifically to the low oxygen tension regions present in tumors. In this work, we develop a mathematical model that describes oxygen transport in a cylindrical annulus of cancerous tissue fed by a single cylindrical capillary. The oxygen transport model was used to evaluate mixtures of red blood cells and six types of HBOCs that consisted of two polymerized hemoglobins, two liposome-encapsulated hemoglobins, and two hydrogel-encapsulated hemoglobins. These HBOCs span a wide range of oxygen affinities (P(50)s) ranging from 5 to 54 mm Hg to study the effect of P(50) on the ability to target oxygen delivery to hypoxic regions of a tumor and hence improve tumor oxygenation. The results of these simulations indicate that each HBOC has an effective pO(2) range in which it unloads the most oxygen, which is dependent on the HBOC's P(50). Although it would seem that the HBOC with the lowest P(50) (5 mm Hg) should be the best option for O(2) delivery to hypoxic tumors, our results indicate that when this is the case, the lowest P(50) HBOC yielded insufficient O(2) delivery to normoxic tissues (inlet pO(2) approximately 95 mm Hg). Because HBOCs would be transfused into a patient's blood stream before initiating a particular cancer therapy, sufficient oxygen must be delivered to both normoxic and cancerous tissues to maintain normal tissue functions when oxygenating the tumor. Human blood supplemented with a hydrogel-encapsulated Hb with a P(50) of 10 mm Hg was found to deliver sufficient oxygen to normoxic tissue while increasing oxygen delivery to hypoxic regions of tumorgenic tissue. Taken together, this work represents a novel strategy for enhancing the efficacy of existing cancer therapies.

Conjugation of methoxypolyethylene glycol to the surface of bovine red blood cells.

Methoxypolyethylene glycol (mPEG) covalently bound to the surface of human red blood cells (hRBCs) has been shown to decrease immunological recognition of hRBC surface antigens (Bradley et al., 2002). However, there is an increasing shortage of hRBC donations, thus making hRBCs scarce and expensive (Davey, 2004; Riess, 2001). The goal of this study is to similarly PEGylate the surface of bovine RBCs (bRBCs) with the aim of reducing the demand on human blood donations needed for blood transfusions. This study investigates the feasibility of modifying the surface of bRBCs with the succinimidyl ester of methoxypolyethylene glycol propionic acid (SPA-mPEG) for use as a potential blood substitute. The oxygen binding affinity of PEGylated bRBCs was moderately increased with increasing initial SPA-mPEG concentrations up to 4 mM when reacted with bRBCs at a hematocrit of 12%. Oxygen transport simulations verified that SPA-mPEG conjugated bRBCs could still transport oxygen to pancreatic islet tissues even under extreme conditions. PEGylated bRBCs reconstituted to a hematocrit of 40% exhibited viscosities on the order of approximately 3 cp, similar to hRBCs at the same hematocrit. Taken together, the results of this study demonstrate the success of PEGylating bRBCs to yield modified cells with oxygen binding, transport and flow properties similar to that of hRBCs.