TISSUE ENGINEERING Volume 12, Number 2, 2006 © Mary Ann Liebert, Inc.
Photosynthetic Oxygen Generator for Bioartificial Pancreas KONSTANTIN BLOCH, Ph.D.,1 ELI PAPISMEDOV, M.Sc.,1,2 KARINA YAVRIYANTS, B. Sc.,3 MARINA VOROBEYCHIK, M.Sc.,1 SVEN BEER, Ph.D.,2 and PNINA VARDI, M.D.1
ABSTRACT Immunoisolation of pancreatic islets interrupts their vascular connections and results in severe cell hypoxia and dysfunction. This process is believed to be the major obstacle to a successful cure of diabetes by implantation of bioartificial pancreas. Here we describe a new technology for microalgabased, photosynthetic oxygen supply to encapsulated islets, in which a thermophylic strain of the unicellular alga Chlorella was used as a natural photosynthetic oxygen generator. Following determinations of the optimal number of alga cells required for compensation of islet respiration, an appropriate number of islets and algae were co-encapsulated in alginate and perifused with oxygenfree medium at increasing glucose concentrations. No insulin response to glucose was obtained in islets alone, or upon inactivation of photosynthesis by darkness. However, under illumination, photosynthetic-dependent oxygen generation induced higher glucose-stimulated insulin response when compared to normoxic perifusion. Such photosynthetic oxygen generation may have a potential application in development of various bioartificial tissues, in particular the endocrine pancreas.
INTRODUCTION
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HE RECENT SUCCESS OF THE Edmonton protocol for transplanting pancreatic islets to patients with type 1 diabetes opened up new perspectives in the treatment of this worldwide disease.1 However, the potential development of severe side effects associated with nonselective anti-rejection, immunosuppressive therapy indicates the need for different, safer, therapeutic solutions, such as offered by transplantation of immunoisolated cells. Selective islet immune protection is achieved by their encapsulation in semipermeable matrixes, such as alginate,2,3 or implantation of islets in solid devices surrounded by diffusion membranes.4–6 However, immunoisolation leads to total loss of the vasculature, resulting in substantially decreased delivery of oxygen and nutrients to islet cells. Since endogenous pancreatic islets have an extremely dense, glomerular-like angioarchitec-
ture, the interruption of their vascular connection results in serious functional abnormalities and cell death.7,8 Limitations in oxygen diffusion leads to establishment of severe hypoxic conditions, with a decrease in stimulated insulin secretion and subsequent cell death.9,10 Recently, several new approaches to overcome oxygen transport limitations of immunoisolated islets have been proposed, including prevascularization of the diffusion membrane, after exposure to angiogenic factors,11 and introduction of PEG-conjugated hemoglobin as an oxygen carrier to facilitate its transport to microencapsulated islets.12,13 To provide oxygen independently from the oxygen tension in the surrounding tissue, an oxygen generator based on electrolysis of water to form oxygen and hydrogen was also developed and introduced into an immunobarrier diffusion chamber containing insulin-producing cells.14 Here we explore a novel technique for oxygen provision to immunoisolated islets, which adopts the photosynthetic
1Diabetes and Obesity Research Laboratory, Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel-Aviv University, Beilinson Campus, Petah Tikva, Israel. 2Department of Plant Sciences, Tel-Aviv University, Tel-Aviv, Israel. 3Beta-O Technologies, Ltd., Petah Tikva, Israel. 2
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capacity of algae to generate oxygen while utilizing CO2 in the process. For this purpose, a green unicellular thermophylic Chlorella sorokiniana strain was used as a natural photosynthetic oxygen generator to support stimulated insulin secretion from encapsulated pancreatic islets. To test the feasibility of algal-based oxygen generation to support insulin secretion of pancreatic islets in an anoxic environment, alginate capsules containing entrapped pancreatic islets and unicellular algae were perifused with oxygen-free medium. The system was adapted for determination of insulin secretion, oxygen consumption, and photosynthetic efficiency. We found that alga-based photosynthetic oxygen production can compensate oxygen consumption of islets and provide optimal insulin secretion from encapsulated islets perifused with oxygen-free medium. Such a technology may have a potential application for cell therapy and for other medical fields where local improvement of oxygen tension is required.
MATERIALS AND METHODS Islet isolation and cell culture Islets were isolated from the pancreas of 25 g male ICR mice by the collagenase digestion technique.15 Before encapsulation and perifusion, the islets were cultivated overnight in a CO2 incubator in RPMI 1640 media supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 g/mL streptomycin. A mouse insulinoma cell line (TC-tet) was cultivated as described previously.16
Algal culture An axenic strain of Chlorella sorokiniana, with an optimal temperature of 37°C, was purchased from UTEX (Catalog no. 1230, Austin, TX). In our laboratory, the stock cultures were cultivated in Proteose medium (UTEX) in a CO2 incubator (95% air and 5% CO2) at 37°C and a 16 h photoperiod at an irradiance of 50 mol photons m2 s1.
Islet and algal encapsulation Encapsulation was performed using the modified method of Lim et al.17 Briefly, islets alone or with algal cells were suspended in 1.8% (w/v) sodium alginate LVG (Pronova Biomedical, Oslo, Norway). The mixture was then extruded through a pipette tip to create capsules of 3 mm diameter. The capsules were collected in a 1.5% calcium chloride solution supplemented with 13 mM Hepes, and then sequentially washed with a 0.75 and 0.35% calcium chloride and 0.9% saline solution. Freshly encapsulated islets and algae were used for the perifusion studies. Each capsule contained 23 5 islets and 150–200 103 alga cells. Figure 1 illustrates encapsulated islets alone and islets co-encapsulated with algae.
Oxygen consumption and production The rate of islet/insulinoma cell oxygen consumption and algal oxygen production was measured in a stirred water-jacketed chamber at 37°C using a Clark type oxygen electrode assembly (Hansatech, Norfolk, United Kingdom). One hundred islets or 106 insulinoma cells and/or various concentrations of algal cells resuspended in KRBB supplemented with 16.7 mM glucose were placed in the 1 mL Clark electrode chamber, and changes in the oxygen levels were directly recorded by a computer.
Perifusion system and perifusion protocol The perifusion system consisted of a 1 mL glass-made perifusion chamber (10 10 mm) equipped with a magnetic stir bar in the same Clark electrode setup as described above (Fig. 2). Tubing (TYGON R-3603, ColeParmer, Vernon Hills, IL) entered and exited the chamber through small holes in the glass wall. A peristaltic pump (Ismatec, Glattbrugg, Switzerland) provided a perifusion flow of 0.3 mL/min, and samples for insulin and glucose determinations were taken every 5 min in a fraction collector (RediFrac, Pharmacia Biotech, Bjorkgatan, Sweden). To achieve anoxia, normoxia, and hyperoxia inside the chamber, the perifusion solution was equlibrated with 95% N2 5% CO2 (oxygen was not detectable); 95% air 5% CO2 (pO2 140 mmHg) and 95% O2 5% CO2 (pO2 500 mmHg), respectively. An optical fiber was used for illuminating the perifusion chamber. The perifusion system was placed in a temperature-regulated plastic box at 37°C. Encapsulated islets and algae underwent a pre-perifusion period for 30 min in KRBB containing 5.6 mM glucose and 0.25% BSA. Basal insulin secretion was estimated at the same medium and then switched to KRBB containing 16.7 mM glucose. The level of insulin in samples was estimated using a radioimmunoassay kit (INSIK-5, DiaSorin, Saluggia, Italy), which shows 100% specificity to rat insulin. Glucose levels in the perifusate was determined by the glucose oxidase assay (GTR-P, Sigma, St. Louis, MO). Light intensities were measured with a quantum sensor (Li-Cor, Lincoln, NB).
Statistical analysis The results are presented as means SE. Groups of data were compared using unpaired Student’s t test. Differences were considered significant if p 0.05.
RESULTS Photosynthesis-based oxygen generation as support for islet respiration Using a Clark-type electrode assembly, we found that the oxygen consumption of mouse islets used in our ex-
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FIG. 1. Pancreatic islets and algae entrapped in alginate capsules. (A) Islets only. (B) Islets co-encapsulated with algae. Scale bar, 150 m
periment was 11.9 2.3 pmol/min/islet at 16.7 mM glucose. Mouse insulinoma TC-tet cells served as control group and showed a respiration rate of 1.4 0.3 pmol O2/min/103 cells. Considering that one islet contains about 0.5–1.0 103 cells, the respiration rate of the insulinoma cells was found to be significantly lower than that of the islet cells used in our experiments. The determination of the oxygen consumption rate was used for the calculation of oxygen demand by the pancreatic islets, to be later supplemented by the photosynthetic process during the perifusion experiments. The planned experiment required a nontoxic alga that maintains its photosynthetic capacity in liquid environments at body temperature, survives in close proximity to mammalian cells, and produces sufficient amounts of oxygen when exposed to light. Such requirements were met by the thermophylic strain of Chlorella sorokiniana, an axenic unicellular alga with a cell diameter of 1 m.
These algae were able to grow and photosynthesize in Krebs-Ringer bicarbonate buffer (KRBB), a commonly used medium for islet perifusion studies. Figure 3A shows oxygen production by this alga as a function of irradiance, reaching a maximal level at about 360 mol photon m2 s1 with a plateau response up to 700 mol photon m2 s1. When oxygen production was estimated as a function of algal density (Fig. 3B), a direct correlation (R 0.996) was found between 5 106 to 20 106 cell/mL. The optimal algal concentration and irradiance necessary to maintain a sufficient oxygen supply for islet respiration was calculated according to several parameters likely to influence the photosynthetic process. Firstly, we found that the respiration rate of one islet was approximately 12 pmol/min. Secondly, we took into account that the rate of photosynthetic oxygen production should be equal to islet oxygen consumption. Using the equation
37C Fiberoptic light FIG. 2. Schematic of the perifusion system. The peristaltic pump delivers solutions with different oxygen tensions to the perifusion chamber, equipped with magnetic stirrer and Clark-type oxygen electrode. The fraction collector harvests outflow solutions for insulin and glucose determinations. A fiber optic light source illuminates the perifusion chamber.
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creased to about 40,000 cells. Similar experiments performed with insulin-producing TC-tet cells demonstrate that about 1200 alga cells are required to compensate the respiration of 1000 cells at saturated irradiance, and significantly more at 50 mol photon m2 s1 ( Fig. 4B).
Effect of oxygen level on insulin response of encapsulated islets
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To study the effect of oxygen tension on encapsulated islets, we used a perifusion system for a time-dependent estimation of insulin response to secretagogues. High oxygen tension, normoxia, and anoxia in the chamber were achieved by pumping KRBB supplemented with secretagogues through the perifusion system after pre-
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FIG. 3. Effects of irradiance (A) and cell density (B) on photosynthetic oxygen production by cells of the alga Chlorella sorokiniana. (A) 40 106 cells were loaded in the chamber and illuminated with different irradiances: 0–700 mol photons m2 s1 (n 5). (B) Different concentrations of algal cells (5-20 106 cell/mL) were loaded in the chamber and illuminated at a irradiance of 360 mol photons m2 s1 (n 6). Data points show means; error bars show se.
presented in Figure 3, we found that the oxygen liberated from one algal cell was 0.0009 pmol/min, and, thus, about 13,000 cells were expected to compensate for the respiration of one islet at saturating light (12 pmol/min/ islet:0.0009 pmol/min/alga cell). Using similar calculations, we estimated that the photosynthetic activity of 1500 algal cells would be sufficient to compensate for the respiration of 1000 insulinoma cells. Our experimental results (Fig. 4A), based on direct determination of alga-based compensation of islet oxygen consumption, indicate that at saturated irradiance, the number of algal cells required to compensate for the respiration rate of one islet is about 11,000. This value is very close to that obtained by calculations based on indirect estimations of oxygen consumption and production. When photosynthetic oxygen production was reduced by decreasing the irradiance to 50 mol photon m2 s1, the algal density required to compensate for the respiration of one islet in-
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FIG. 4. Effects of algal density and irradiance on compensation of islets (A) and TC-tet cells (B) oxygen consumption. The glass-made transparent Clark oxygen electrode chamber containing free-floating islets/TC-tet cells and algae was illuminated at 360 mol photons m2 s1 () and 50 mol photons m2 s1 (). Points crossing the dotted line indicate number of algal cells able to compensate pancreatic cells’ oxygen consumption at the two different irradiances.
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a A 5.6 mM glucose
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FIG. 5. Stimulated insulin response from encapsulated islets in the perfusion system at different oxygen tensions. Anoxia (), n 5; normoxia (), n 3, and hyperoxia (), n 4. (A) Effect of perifusion time. Dotted line indicates the shift from low to high glucose concentration in the perifusion chamber. (B) Effect of increasing glucose concentration in the perifusion chamber during a 0 to 75 min perifusion period. Results are expressed as fold stimulation compared to 5.6 mM glucose. R2squared value.
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equilibration with an appropriate gas mixture. Figure 5A illustrates the effect of oxygen tension on stimulated insulin secretion from encapsulated islets. Since significant variations in the basal insulin secretion rate (from 2 to 16 U/mL) were found between different batches of isolated islets, the results were presented as fold stimulation compared to the basal insulin secretion at 5.6 mM glucose. An increased insulin response to secretagogues at both normoxic (140 mmHg) and hyperoxic conditions (500 mmHg) was found. Nevertheless, no significant differences in insulin response between pancreatic islets perifused at high oxygen tension and normoxic conditions were observed, suggesting an unchanged insulin profile during conditions of normoxia and hyperoxia (Fig. 5B). These data show that encapsulated mouse islets, at least for some time, preserve their normal range of functional activity at relatively high oxygen tension. However, the inner oxygen concentrations of the capsule may be significantly lower due to diffusion limitations, leading to a restricted oxygen supply to the central part of the capsules. This would lead to poorer insulin response from encapsulated islets when compared to well-oxygenated free-floating islets, as has been shown by various au-
thors.12,18 In our experiments, perifused encapsulated islets showed a complete inhibition of stimulated insulin response during anoxia, unlike the basal rate of insulin secretion which was not affected (Fig. 5). These results are consistent with previous reports, which showed that only the stimulated insulin secretion is affected during severe hypoxia and anoxia, probably due to greater depletion of ATP-based energy stores in pancreatic islets.10,19
Photosynthetic oxygen support of stimulated insulin secretion in an anoxic environment To explore the possibility that algal-based photosynthetic oxygen generation may support the islets’ functional activity in oxygen-free environments, we studied the stimulated insulin secretion of islets co-encapsulated with illuminated Chlorella sorokiniana cells during perifusion at anoxic conditions. Figure 6 shows the insulin response of encapsulated mouse pancreatic islets alone, and islets co-encapsulated with algae. The average number of islets per capsule was 23 5, and that of co-entrapped algal cells was about 150–200 103. According to our experimental results, such an algal concentration
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FIG. 6. Stimulated insulin responses from islets co-encapsulated with algae and perifused with anoxic media. (A) Effect of perifusion time during the illumination period (n 5). (B) Effect of glucose concentration in the perifusion chamber during a 0 to 75 min illumination period (n 5). (C) Effect of illumination and darkness periods. Thin lines show periods of illumination; bold lines indicate period of darkness. The data present two independent experiments. The vertical dotted line indicates a shift from low to high glucose concentration in perifusion chamber. Islets co-encapsulated with algae (), encapsulated islets (). Results are expressed as fold stimulation compared to 5.6 mM glucose.
at saturating irradiances was sufficient to compensate for the respiratory need of the encapsulated islets. At this cell density, oxygen produced by the algae is bound to diffuse in close contact to adjacent islets, providing an efficient oxygen supply to the insulin-producing cells. Data presented in Figure 6A demonstrate that during illumination, islets co-encapsulated with alga cells and placed in an anoxic environment show a statistically significant (p 0.01) increase in insulin secretion in response to secretogogues, in contrast to encapsulated islets alone. Interestingly, the insulin response to elevated glucose concentration obtained during photosynthesis-dependent islet respiration (Fig. 6A) was higher than that obtained by islets perifused at normoxic conditions (Fig. 5A). Despite the strong association between the increasing glucose concentrations in the perifusion chamber and the insulin response of islets co-encapsulated with algae, during the light phase (Fig. 6B), no oxygen was detected in
the perifusate, suggesting that most of the oxygen generated during the photosynthesis was consumed by the islets’ respiratory process. When the experiments were repeated during a dark phase, no stimulated insulin secretion was detected (data not shown), confirming the ability of light-induced photosynthetic oxygen generation to support adequate insulin secretion. Further evidence supporting the efficiency of alga-based oxygen generation for islet function was obtained from photosynthesis switch on/off experiments, which induced a rapid inactivation/activation of the photosynthetic process. When photosynthesis was inactivated in the dark phase, the oxygen tension in the capsule began to decline due to both islet and algal respiration. Figure 6C shows the reduction of stimulated insulin secretion during darkness in a timedependent manner: about a 50% reduction of maximal insulin response was observed after 30 min of darkness, while 50 min of darkness induced a total inhibition of the
PHOTOSYNTHETIC OXYGEN GENERATOR FOR BIOARTIFICIAL PANCREAS stimulated insulin response. Re-illumination of the system did not restore the original insulin secretion level, but caused a new level of secretion which was higher than the initial one. Such a cell response implies that recovery of beta cell function from prolonged periods of anoxia is not immediate and may not be complete, and longer period of cell oxygeneation may be needed to adequately repair the anoxia-induced cell injury, if at all.
DISCUSSION The pancreatic islets are known to be highly susceptible to insufficient oxygenation. Immunoisolation of islets interrupts their vascular connections, resulting in severe hypoxia and cell dysfunction, which are believed to be the major obstacle to a successful cure of diabetes by implantation of a bioartificial pancreas. The rate of oxygen consumption varies among different insulin-producing cells and according to cell respiratory activity. It was found to be 1.5–4.2 pmol/min in mouse pancreatic islet20–22 and 5–12 pmol/min for rat islet23,24 and increased during dynamic islet respiration induced by elevated glucose concentrations.23,25 A slightly lower oxygen consumption rate was found in mouse TC3 insulinoma cells (1.5 pmol/min/103 cells) at basal glucose level (5.5 mM), with no increase in oxygen demand during higher glucose-stimulated insulin secretion. In such tumoral cells, no difference in oxygen consumption rate was found between encapsulated and nonencapsulated TC3 cells.26 Both the oxygen diffusion limitation and oxygen consumption of pancreatic islets play a crucial role in the development of oxygen depletion in encapsulated islets. In the present study, we used a known technology of cell encapsulation in alginate as a model for islet immunoisolation. To imitate an oxygen-concentration gradient inside the immunoisolated sphere, islets were entrapped in macrocapsules of 3 mm diameter. Insulin secretion from encapsulated pancreatic islets is known to be very sensitive to depletion in oxygen tension, resulting in cell dysfunction and necrosis after transplantation. Both, high islet oxygen consumption and poor diffusion properties of oxygen through the alginate are believed to be responsible to such islet failure.10,27 Here we report for the first time that a relatively small number of photosynthetic microalga Chlorella sorokiniana cells are capable of supplying a relevant concentration of oxygen for respiration and function of islets located in vitro anoxic environments. Green unicellular algae are anciently evolved organisms equipped with a powerful photosynthetic system. In nature, they use sunlight energy to drive the synthesis of organic molecules from CO2 and water and liberate oxygen as a byproduct. The remarkable adaptation capacity of algae led to the natural selection of a large number of species capable of surviving in various extreme environments.28 In addition,
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microalgae are capable of creating symbiotic consortium with corals in nature29 and with other oxygen-consuming microorganisms in laboratory conditions.30,31 Because the algal diameter is about 10 times smaller than the diameter of a pancreatic beta cell, the total mass of algal cells required to compensate for the islet respiration could be relatively small and equal to the total volume of encapsulated islets. In addition, our data showed that the insulin response to elevated glucose concentration obtained during photosynthesis-dependent islet respiration was higher than that obtained by islets perifused at normoxic conditions. This phenomenon may suggest that, firstly, a relatively low oxygen diffusion into the capsules at normoxic perifusion is insufficient to achieve the optimal islet insulin production/secretion capacity and, secondly, results in the formation of central hypoxic zone in the capsule with lower insulin response. In contrast, the oxygen diffusion from illuminated algae was expected to be homogeneous in both the central and peripheral zones of the capsule, enabling optimal insulin response of most islets. Photosynthetic oxygen generation may be utilized in the development of various bioartificial tissue transplants, in particular the endocrine pancreas, as support for insulin secretion in oxygen-deficient conditions. Recent progress in the fields of light transmission and microelectronics would allow for the optimization of such systems with new light-emitting diodes as an efficient energy supply to the photosynthetic oxygen generator. Further investigations should be made to estimate the effects of long-term co-encapsulation on the functional activity of islets and algal-based photosynthetic oxygen production. Many issues related to optimization of islet-alga associations, such as immune rejection, mass transfer limitations due to fibrosis, matrix stability, adequate illumination, and energy supply, should be solved before this approach can be used for the construction of long-term functioning bioartificial pancreas. Alternative approaches may be based on the creation of a bioartificial pancreas containing two retrievable compartments–one with islets and one with photosynthetic algae, separated with a gas-permeable membrane for oxygen and carbon dioxide exchange between the mammalian and plant cells.
ACKNOWLEDGMENTS This study was partially supported by Beta-O2 Technologies, Ltd. We thank Shimon Efrat for providing TCtet cells.
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