An In Vitro System to Evaluate the Effects of Ischemia on Survival of Cells Used for Cell Therapy
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- Davis, B.H., Schroeder, T., Yarmolenko, P.S. et al. Ann Biomed Eng (2007) 35: 1414. doi:10.1007/s10439-007-9301-2
Maintaining cell viability is a major challenge associated with transplanting cells into ischemic myocardium to restore function. A likely contributor to significant cell death during cardiac cell therapy is hypoxia/anoxia. We developed a system that enabled quantification and association of cell survival with oxygen and nutrient values within in vitro constructs. Myoblasts were suspended in 2% collagen gels in 1 cm diameter × 1 cm deep constructs. At 48 ± 3 h post-seeding, oxygen levels were measured using microelectrodes and gels were snap-frozen. Bioluminescence metabolite imaging and TUNEL staining were performed on cryosections. Oxygen and glucose consumption and lactate production rates were calculated by fitting data to Fick’s second law of diffusion with Michaelis–Menten kinetics. Oxygen levels dropped to 0 mmHg and glucose levels dropped from 4.28 to 3.18 mM within the first 2000 μm of construct depth. Cell viability dropped to approximately 40% over that same distance and continued to drop further into the construct. We believe this system provides a reproducible and controllable test bed to compare survival, proliferation, and phenotype of various cell inputs (e.g., myoblasts, mesenchymal stem cells, and cardiac stem cells) and the impact of different treatment regimens on the likelihood of survival of transplanted cells.
KeywordsMyoblast Ischemia Stem cell Cardiomyoplasty Myocardial infarction
The use of adult stem and progenitor cells to repair pathophysiological consequences of myocardial infarction in the human heart12,15,17,20,22,31,37,41,43,51 has become widespread. However, a major obstacle with this technology is the failure of cells to survive long term.29,35,48 Previous studies suggest that more than 70% of the cells injected during cellular cardiomyoplasty die within the first 3–4 days after transplantation.1,26,40,46,53 While the exact mechanisms responsible for cell death are unknown, it is hypothesized that the ischemic microenvironment surrounding injected cells is a major contributor to the poor survival rate.30,54 However, very few researchers have directly examined the role of ischemia on the survivability of transplanted myoblasts, mesenchymal stem cells, or bone marrow mononuclear cells.26,42 One reason for this limited research is that determining the impact of ischemia and low-nutrient concentration, per se, on survival of transplanted cells is difficult to distinguish in an animal model, especially in the face of impacts from inflammatory cells, cytokines, and matrix metalloproteases, to name a few. Because of these in vivo complexities, there is a need to create an in vitro model system to directly evaluate the impact of ischemia on the survival of cells being transplanted.
In these types of studies, an in vitro model system holds several advantages over in vivo animal models. First, in vitro models enable control of environmental factors such as inflammation, cell rejection, cytokines, etc., on transplanted cell survival, thereby isolating the role of ischemia. Second, the use of an in vitro model allows easy and reproducible changes in the availability of oxygen and nutrients to the cells, permitting a more accurate determination of the thresholds of oxygen or nutrients a particular cell type needs to survive. Finally, in vitro oxygen and nutrient consumption rates of the transplantable cells are easily quantified within a model system, whereas accurate measurement of in vivo metabolism is difficult to discern from that of native, host cell metabolism. The ability to measure these consumption rates is crucial to understand the role of ischemia on cell death because injected cells are competing against each other as well as host cells, for limited oxygen and nutrients in the ischemic/hypoxic infarct. By understanding the metabolic demands of the injected cells, we can potentially calculate the levels of glucose and oxygen available to cells injected into myocardial infarct scar. With better estimates of these oxygen and nutrient values and viability of cells at those values (measured in vitro), it should be possible to devise injection strategies designed to maximize transplanted cell survival. Further, precise calculation of oxygen and nutrient consumption rates of these cells under the gradient of conditions experienced in 3-D constructs or infarct should provide valuable information for computational models designed to predict the viability of cells transplanted in vivo in both animals and in patients. To date, a limitation of many in silico analyses of non-cardiac cells transplanted into a cardiac milieu is that they utilize only approximations of cell oxygen and glucose consumption rates (OCR and GCR) and lactate production rates (LPRs) because few, if any, reliable measurements exist quantifying these parameters in an infarct, or infarct-like, ischemic milieu.
Although little data exist on the specific oxygen and nutrient metabolism and viability of progenitor cells, OCR and GCR of various other cell types have been examined, both in vivo and in vitro in non-cardiac tissues. The current gold-standard technique to quantify oxygen levels and consumption rates in tissues or constructs uses probes, such as recessed-tip glass microelectrodes,38 placed into tissues, constructs, or into the media of cell culture vessels.27,36,50 Microelectrodes were used to monitor oxygen in the current study. Glucose and other metabolites can be measured in a number of ways, but a new, potentially powerful technique that shows promise in tissues and 3-D cultures is bioluminescence metabolite imaging (BMI).32 BMI involves combining frozen sections of the tissue of interest with an enzyme solution that links lactate or glucose to a light producing luciferin–luciferase system. This technique has been previously used to examine tissues as diverse as tumors39,49 and hearts.32 Finally, nitro blue tetrazolium chloride (NBT) and Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) have been used extensively, in histology sections, to examine cell viability and apoptosis, respectively.18,19,21,33,34,52
The goal of this project was to develop an in vitro system to quantify the effects of ischemia on the survival of transplanted cells by seeding porcine myoblasts in type I collagen constructs. This system enabled manipulation of individual parameters to evaluate their impact on cell survival in an environment with hypoxia and glucose conditions encompassing those found in myocardial infarct scar. The 3-D collagen gel model system was designed such that oxygen and nutrient levels were controlled at the surface of the gel and diffusive transport, coupled with oxygen and nutrient consumption by the cells, enabled a gradient of nutrients and oxygen to exist throughout the construct: this gradient was designed to encompass values measured in infarcted pig heart (manuscript in progress). The constructs were cultured for 48 h, with media changed every 12 h, to allow the constructs to reach steady-state. Recessed-tip oxygen microelectrodes were used to measure oxygen gradients23 and bioluminescence imaging was used to measure the gradient of glucose and lactate through the gels. Cell viability and apoptosis were measured using NBT and TUNEL staining, respectively. OCR and GCR were calculated using a custom algorithm written in MATLAB.
Skeletal Muscle Cell Preparation
A 500–700 mg biopsy of the semi-membranosus skeletal muscle was obtained from the hindlimb of swine under general anesthesia (3% isoflurane) (n = 6 pigs). The tissue was mechanically dissected into 1 mm3 pieces and washed twice with Dulbecco’s phosphate buffered saline (PBS), as previously described by our group.45 The tissue was then plated in growth medium containing 10% fetal bovine serum and 0.5% gentamicin (10 mg/mL). After 3 days, tissue fragments were triturated, and additional growth media was added. Fragments were removed after 5 days. The myoblasts were expanded a total of 28 days post-biopsy, 6–8 passages, while maintaining less than 70% confluence. This technique is used regularly in our laboratory to maintain greater than 80% desmin positive myoblasts.43
Gel constructs, 1 cm diameter × 1 cm depth, were composed of myoblasts suspended in type-I collagen, the primary component of myocardial infarct scar. Creation of collagen gel constructs required 4× concentrated Dulbecco’s modified eagle’s medium (DMEM) to achieve the desired initial glucose concentration within the construct. The 4× DMEM was obtained via addition of double distilled H2O to low glucose DMEM powder (Invitrogen, Carlsbad, CA, catalog # 31600-034). A 2 mg/mL collagen mixture was prepared on ice by combining high concentration rat tail collagen type I (BD Biosciences, San Jose, CA, catalog # 354249) with 4× DMEM, and NaOH was added to neutralize pH to 7.0.
Cells were trypsinized, washed twice in PBS, and re-suspended in growth medium. The cell solution was then mixed with the collagen mixture to obtain a final concentration of 2 million cells/mL gel. This initial concentration lead to a final concentration of approximately 2.5 million cells/mL in the gels at 48 ± 3 h, the time chosen for measurement in order to obtain pseudo-steady state conditions. After mixing, the gel solution was pipetted into a well of a standard 24 well tissue culture plate (Falcon, BD Biosciences, San Jose, CA, catalog # 353047). Gels were placed in a standard tissue culture incubator for 45 min to solidify. Growth media was then added (500 μL) to the top of the gel. Growth media was replaced every 12 h for 48 ± 3 h. For quantification of cell viability, a subset of gels had growth media containing 0.05% NBT for 6–8 h prior to snap freezing. As an extra perturbation to the system, after gel formation, a set of constructs (n = 3) were placed in a tissue culture incubator maintained at 5% oxygen for the 48 h preceding measurements.
The oxygen microelectrode was introduced into the collagen gel and positioned at a depth of 3000 μm from the gel surface using a micromanipulator (Model MO-102, Narishige International, East Meadow, NY). At this position, the microelectrode output corresponded with an oxygen tension of 0 mmHg. The system was then allowed to stabilize for 15 min. While continuously recording, the microelectrode was then retracted towards the surface at 50 μm intervals every 60 s. This process was repeated three times per gel (n = 6 gels). For measurements from gels subjected to hypoxia during the system perturbation (n = 3 gels), the micromanipulator, culture plates, and waterbath were enclosed by a plastic container. Oxygen tension within the chamber was maintained at 5% oxygen by use of a ProOx hypoxia chamber (BioSpherix Ltd., Redfield, NY) during measurements.
Cell Viability (TUNEL + NBT)
A subset of gels (n = 3 control and n = 3 hypoxia) was exposed to 0.05% NBT (Molecular Probes, Invitrogen, Carlsbad, CA, catalog # N-6495) growth medium for 6–8 h prior to snap freezing. NBT is cell-permeable and reduces to a blue formazan product by viable cells. Therefore, blue-colored cells were considered viable at the time of snap freezing and uncolored cells were considered dead. To measure the contribution of apoptosis to cell death, TUNEL analysis was performed on cryosections (Roche Applied Sciences, Indianapolis, IN, catalog # 11684817910). A propidium iodide (PI) secondary stain provided verification of the total number of cells from all sections. A custom MATLAB algorithm was written to count the total number of cells (PI positive), live cells (NBT positive), and apoptotic cells (TUNEL positive) across each gel cryosection. A total of 10, non-overlapping regions on 4–5 cryosections of each gel were examined.
Bioluminescence Metabolite Imaging
For bioluminescence imaging, cryosections were freeze-dried in a Labconoco Freeze Dry System (Labconoco Co., Kansas City, MO) and stored at −80 °C. Imaging was performed as described previously.32 In brief, successive central sections of the gels were inverted and placed in a chamber filled with a solution linking the substrate of interest (glucose or lactate) to light emission through a bacterial luciferase.50 Data were acquired on a Zeiss Axioskip 2 microscope in a black box with an Andor Technology DV465C-FI CCD camera. Images, at 15× magnification (resulting in pixels dimensions of 15 × 23 μm), were accumulated through photon counting of the emitted light. Carefully controlled glucose and lactate standards were also acquired using the same procedure. Calibration with these standards allowed conversion of emitted light intensity to the associated metabolite concentration. A custom MATLAB (The Math Works Inc., Natick, MA) algorithm was then used to find the average pixel intensity and convert it to the average concentration of the metabolite of interest at each depth from the surface of the gel (n = 3 images per gel). The total number of pixels used to determine each average value per depth (approximately 400 pixels per value) varied depending on the image, as bubbles were excluded from the analysis. The analysis averaged together all ‘relevant’ (identified as within the gel but without a bubble) pixels for each 100 ± 10 μm depth. Results were plotted as average intensity of three bioluminescence images per gel vs. depth for later analysis regarding glucose consumption and lactate production.
Calculating Oxygen and Glucose Consumption
Data are expressed as the mean ± standard error of the mean. To assess the success of the curve-fitting algorithms to calculate consumption and/or production terms, R2 values and the root mean squared (RMS) of the residuals of the fit were calculated. Comparisons of oxygen, glucose, and lactate levels and consumption (or production) with gel depth were performed using one-way ANOVA with Fisher’s PLSD post-hoc analysis between individuals, when ANOVA showed significance. Significance was set at p < 0.05. A two-sample t-test of correlation coefficient with a Fisher’s z-transformation was used to test whether staining of NBT and TUNEL were similarly correlated with depth into the gel constructs. To test the success of the hypoxia system perturbation, differences in oxygen levels, oxygen consumption rate, and viability vs. depth were compared in control and hypoxia constructs using two-way ANOVA. When significance (p < 0.05) was found, Fisher’s PLSD test was used to find pairwise differences. When significance of interaction coefficient allowed, oxygen values and consumption rates and viability were split by depth to determine differences between control and hypoxia results at each depth.
Cell Viability Results
Curve fitting for oxygen consumption resulted in an R2 of 0.994 and an RMS of the residuals less than 2% of the maximum oxygen level. For glucose consumption, curve fitting of control conditions resulted in an R2 of 0.897 and an RMS of residuals of less than 4.5% of the maximum glucose levels. Finally, for lactate production calculations, curve fitting resulted in an R2 of 0.902 and an RMS of the residuals less than 4% of the maximum lactate level. The curve fit to the viability data had an R2 of 0.934 for NBT data and 0.862 for TUNEL data. For the system perturbation, the curve fit for oxygen data had an R2 of 0.928 with an RMS of residuals of less than 7.5% of the maximum.
Oxygen Measurements and Oxygen Consumption Rate (OCR)
Glucose Levels and Glucose Consumption Rate and Lactate Levels and Lactate Production Rate
The combined data show that over 75% of cells died by 3000 μm depth into the constructs. At this depth, mean oxygen levels dropped below 0.1 mmHg, but glucose levels remained above 2 mM.
The goal of this project was to develop an in vitro system to test the role of oxygen and nutrient deprivation on survival of myoblasts, cells portrayed as relatively “ischemia-resistant,” and used for transplantation into ischemic myocardium. To achieve this goal, a number of hurdles present in currently used systems had to be overcome. Previously, cellular oxygen and glucose metabolism was quantified primarily in 2-D culture systems or in stirred cell suspensions. In these studies, oxygen consumption under hypoxia has typically3,10,13 been measured as a difference between the oxygen concentrations at an input and an output of a cell culture area. Similarly, glucose consumption has commonly been examined by measuring the average drop in glucose in media over time.3,10,16 Examination of cell viability and metabolism at low levels of oxygen and glucose is problematic in these systems because these methods are incapable of examining pseudo-steady state conditions that mimic in vivo scenarios. When very low glucose levels were desired, media had to be changed often to maintain a consistent level of glucose to the cells. Unfortunately, media changes are complicated by a desire to keep oxygen levels low in the cell culture system. A flow system could be used, but a filter system must be added to keep floating dying or dead cells within the system to ensure accurate viability measurements. The use of the novel 3-D culture system described in this study overcomes these limitations. This system allows a range of oxygen and glucose levels to be supplied to the cells of interest by cellular metabolism and diffusion. The absolute ranges achievable are then controlled by gel dimensions, cell density, and glucose and oxygen concentrations supplied at the surface of the gel.
After the cells reached pseudo-steady state (48 h post-seeding), we used proven techniques of gold-plated oxygen microelectrode measurements and metabolic bioluminescence imaging, combined with post-hoc modeling and histology, to measure the metabolism and viability of myoblasts. The system was robust, producing consistent data, even at low levels of oxygen and nutrients. The maintenance of residuals of less than 5% of the measured values in the curve-fitting algorithm indicated good accuracy in the curve-fitting algorithm used to calculate OCR, GCR, and LPRs. The OCR of myoblasts measured at the construct surface fit well with values found in the literature.11,28 Further, the oxygen consumption rate of myoblasts remained constant over a wide range of oxygen tension and was reduced only in the presence of very low oxygen levels. The Pm value in the Michaelis–Menten kinetic model for oxygen fit was 2.86 ± 0.88 mmHg, which was up to five times larger than published values for many tissues,9,44 but well within the range of Pm published for myocardial muscle cells.7,8,36 The maintenance of OCR over a range of oxygen tension may indicate the importance of providing adequate oxygen supply to preserve cell viability. Previous papers have shown that low oxygen can stimulate hypoxia inducible factors, such as HIF-1α, which affect metabolism of cells under hypoxia4 and can lead to growth arrest and apoptosis via the p53 pathway.5,14 GCR and LPR rose with increasing distance into the gel. These increases were likely due to a shift toward anaerobic metabolism caused by limited oxygen levels deep within the gels. The three- to five-fold increase in GCR and LPR agree well with increases reported for other mammalian cells under 20% oxygen vs. severe hypoxia.3 The gradient of ischemia achieved within the gel correlated with cell viability at each depth. Therefore, our experimental setup enabled determination of where along the oxygen/nutrient gradient cells begin to die due to ischemic conditions.
This system is also designed to allow side-by-side comparison of a variety of cell types and conditions with potential for cell transplantation. Not only can the system be used to test which cells are “best suited” to survive in ischemic environments similar to infarcted myocardium, but this setup also provides a reproducible and high throughput method to test variables designed to improve the ability of cells to survive ischemic shock. Several conditions can be examined at once, using cells from the same animal, allowing for better evaluation of techniques attempting to increase viability of the transplanted cells. In order to produce a variety of stress conditions, growth media could be replaced with media containing increased or decreased levels of glucose, and the oxygen level in the incubator could be altered to create a high or low oxygen environment at the gel surface. To show this ability to customize parameters, we examined oxygen tension, OCR, and viability of myoblasts within a construct cultured in an incubator maintained at 5% oxygen. As expected, oxygen tension within the construct was significantly reduced (p < 0.05). OCR was similar at the gel surface but was significantly reduced from 1000 to 1500 μm (p < 0.05). Finally, cell viability was significantly reduced within control constructs from the gel surface down to 1700 μm depth (p < 0.05).
The major limitation of the system presented here is the inability to account for all of the complexities of the in vivo infarct milieu. While this setup provides a more specific study of the role of hypoxia and glucose starvation on cell survival, it neglects other potential contributors to cell metabolism and function, such as cytokines and growth factors that could change cell metabolism and eschew our results. A further limitation is that in our setup, cells are evenly distributed throughout the simulated infarct. In animals or patients, however, the distribution is more complicated: cells are injected in pockets with much higher cell density. In these discrete areas of high cell density, oxygen, glucose, and lactate levels could change quickly, potentially changing metabolism over a very short scale. The reason we chose to use evenly distributed cells at low cell density was to provide greater accuracy in our measurements of oxygen and nutrient consumption to validate the system. Likely, determining how results obtained using this simplified ischemic infarct system will impact cell viability in a more complicated in vivo infarct, even ignoring other causes of cell death, will require the use of computer modeling. Ongoing modeling studies utilizing the data obtained here and accounting for multiple injection geometries, injection locations, and cell densities are underway to better understand how ischemia impacts injected cell survival.
Future studies will focus on two areas: a side-by-side comparison of the viability of various cell types used or proposed for cardiac cell therapy—including bone marrow, myoblasts, and cardiac or other tissue derived stem cells—to determine resistance to ischemic cell death followed by studies in which we alter cell or milieu-dependent parameters in an attempt to increase cell survival, such as decreasing the glucose or oxygen consumption rates of the transplanted cells. By creating an in vitro system where we can alter either milieu or cells, we expect to be able to develop ways to improve the survivability of cells in an ischemic environment similar to myocardial infarct scar, and thereby help to maximize the improvements seen following cell therapy for myocardial infarction.
The described 3-D system can be used to measure the metabolism and survival of cells under a variety of oxygen and glucose levels likely encountered after transplantation into infarcted myocardium. Oxygen and glucose levels within the system ranged from values prevalent in uninjured tissue to levels lower than those found in infarcted myocardium. Relevant parameters of the system (e.g., available oxygen tension) could be reproducibly altered and the effect on cell viability measured. Comparison between previously published data and values measured at the surface of our system indicate that the techniques we used to measure oxygen tension, glucose concentration, OCR, GCR, and LPR within the construct were valid. It is our hope that this system will be used to study metabolism and viability of a host of transplantable cell types under a range of conditions in an attempt to improve viability of cells transplanted during cardiac cell therapy.
This work was supported in part by NHLBI/National Institutes of Health awards to Dr. Taylor (R-01 HL-63346, HL-63703). We would also like to thank Robert Nielsen and Zahid N. Rabbani for their help.