Engineering, Columbia University, New York, NY, USA; 3 Departments of Biomedical Engineering and Medicine, Columbia University, New York, NY USA

Transcription

1 Creation of Live Lung Slices and Analysis of Cell-Matrix Interactions for Regionalized Human Lung Regeneration Sneha Subramaniam 1 *, John O'Neill 2, Gordana Vunjak-Novakovic 3 1 Columbia College, Columbia University, New York NY 10027; 2 Department of Biomedical Engineering, Columbia University, New York, NY, USA; 3 Departments of Biomedical Engineering and Medicine, Columbia University, New York, NY USA Abstract Currently, the definitive treatment for end-stage organ failure is orthotropic transplantation, or the transplantation of a whole organ from one patient to another. Due to a critical shortage of viable donor organs, however, the number of unwarranted deaths due to chronic lung disease has increased: an estimated 120,000 deaths per year in the United States alone. Additionally, only about one in five donor lungs are usable for transplantation. In recent years, focus has shifted to regenerative medicine and, specifically, tissue engineering approaches in which cellular sources and scaffolding materials are combined to create functional tissue constructs. Tissue engineering provides a safer alternative to transplantation by minimizing or eliminating most of the aforementioned risks. Thus, the overall objective of this project is to increase the available number of viable lungs by regionally re-conditioning the damaged part(s) of lungs deemed marginally unacceptable for transplant. Re-conditioning includes de-cellularizing damaged or dysfunctional regions of the air space in the lungs Furthermore, some tissue engineering approaches may be incorporated into the patient's own vascular supply for improved blood flow and circulation throughout the implant. The optimal temperature for decellularization and recellularization is 37 o C. However, the ideal temperature for vasculature preservation is 4 o C. Thus, one significant problem that must be investigated is the effect of varying temperature on the processes required for vasculature maintenance and re-cellularization. In addition, new methods must be established that permit micro-scale analysis of live lung tissue, in particular the vasculature, for this and future studies. Thus, the two goals of the present study were to investigate the attachment of a pulmonary epithelial cell type as a function of temperature as well as to establish a method for generating a thin (<500 μm) live lung slice for future functional studies of the vasculature. Metabolic assays and fluorescent staining were conducted on small airway epithelial cells (SAECs) or cells that are found in lung airways that were grown at different temperatures and different coated plastics. The different coats included gelatin, fibronectin, digested ECM, and a tissue culture plastic control. Results indicated that gelatin was the most effective in cell attachment and that 20 o C was the optimal temperature for confluency and cell density for our project's goals. In addition, 12% gelatin perfusion of lung airways was found to be the most effective for the lung slice. Future work includes trying cellular co-cultures and ips cells for cell attachment and immunological study models on the functional live lung live slice. Introduction Currently, the definitive treatment for end-stage organ failure is orthotopic transplantation, or the transplantation of a whole organ from one patient to another. Due to a critical shortage of viable donor organs, however, the number of unwarranted deaths due to chronic lung disease has increased: an estimated 120,000 deaths per year in the United States alone. Additionally, only about one in five donor lungs are usable for transplantation. Those who are fortunate enough to receive a lung transplant also face risks of inflammation, infection, chronic rejection, and increased morbidity due to a lifelong regimen of immunosuppressant therapy (Badylak, 2011). Therefore, in recent years, focus has shifted to regenerative medicine and, specifically, tissue engineering approaches in which cellular sources and scaffolding materials are combined to create functional tissue constructs. Tissue engineering provides a safer alternative to transplantation by minimizing or eliminating most of the Copyright: 2014 The Trustees of Columbia University, Columbia University Libraries, some rights reserved, Subramaniam, et al. Received 1/1/2014. Accepted 3/14/2014. Published 3/14/14. *To whom correspondence should be addressed: Sneha Subramaniam, Columbia University, New York NY 10027, ss4347@columbia.edu. Spring 2014 Volume 8 10

2 aforementioned risks. Furthermore, some tissue engineering approaches may be incorporated into the patient's own vascular supply for improved blood flow and circulation throughout the implant. Recently, biological scaffold materials composed of natural extracellular matrix (ECM) have shown great promise for construction and remodeling of tissues (Badylak, 2011). The ECM is composed of secreted molecules produced by the resident cells of each tissue or organ. Its three dimensional structure varies based upon the tissue and organ from which it is derived. In turn, the ECM may influence the differentiation, migration, and proliferation of resident cells and serve as a medium for signal transfer between cells. The ECM also constantly changes in response to metabolic activity and mechanical stress (Badylak 2002, Badylak 2011, Brown 2009). In recent years, it has been shown that decellularization of donor organs by removal of native cells provides an acellular, three-dimensional ECM scaffold that can be seeded with stem cells or other progenitor and supporting cell types. ECM is preferable over synthetic scaffolds because it maintains native composition and provides the proper microenvironment for seeded cells to differentiate, i.e., specialize, into a desired cell type. The other important component of tissue engineering is a cellular source. As initially unspecialized cells, stem cells can differentiate into specific cell types and carry out their respective function if placed in the proper microenvironment. Recent studies indicate that induced pluripotent stem cells (ipscs) hold great promise for translational applications in tissue engineering. ipscs can be derived from any somatic (non- reproductive) cell type. With the resetting of four key genes, somatic cells can be de- differentiated back into a pluripotent stem cell state. This means that these cells are sent back to a developmentally naive undifferentiated state. ipscs can then be differentiated into specialized cells of any of the three germ layers (Takahashi, 2007). Since four out of five donor lungs are rejected for transplantation, the overall objective of this project is to increase the available number of viable lungs by regionally re-conditioning the damaged part(s) of lungs deemed marginally unacceptable for transplant. Re-conditioning includes de-cellularizing damaged or dysfunctional regions of the air space in the lungs. Each lung is comprised of a main airway (bronchus) with large airways and small airways successively branching up to 28 times. Most distal in the lung hierarchy are air sacs (alveoli), which expand during inspiration. By de- cellularizing the airways, a crucial component of the respiratory system, this project aims to address regionalized damage. In order to re-cellularize an area, a patient's ipscs are seeded in the de-cellularized region of the lung, thereby creating a chimeric lung [Figure 1A]. A chimeric lung is one that contains cells from two different hosts: in this case the donor and the receiving patient. Figure 1: Chimeric lung generated by integrating partially decellularized donor lung and patient ips cells. Thus, the main goals of the project are: (1) to de-cellularize the epithelium of the lung while (2) maintaining and supporting the native vasculature of the donor lung and (3) recellularizing and differentiating patient-derived ips cells into the appropriate airway epithelial cells types. The current work relates to aims (1) and (2). In order to successfully re-condition the pulmonary epithelium while retaining functional vasculature, a balance between conditions which favor decellularization and vascular maintenance must be established. The optimal temperature for decellularization and recellularization is 37oC. However, the ideal temperature for vasculature preservation is 4oC. Thus, one significant problem that must be investigated is the effect of varying temperature on the processes required for vasculature maintenance and re-cellularization. Spring 2014 Volume 8 11

3 New methods must be established that permit micro-scale analysis of live lung tissue, in particular the vasculature, for this and future studies. Thus, the two goals of the present study were to investigate the attachment of a pulmonary epithelial cell type as a function of temperature as well as to establish a method for generating a thin (<500um) live lung slice for future functional studies of the vasculature. The attachment of small airway epithelial cells (SAECs), which line the airways of human lung, on decellularized human lung matrix was investigated at different temperatures, as part of a larger project to regionally repair or recondition lungs rejected for transplant. Temperature was varied to analyze small airway cell attachment and viability in decellularized lung scaffolds. Temperatures were 37oC (body temperature), 4oC (the temperature at which transplanted lungs are maintained during organ transport), 20 C (room temperature serving as a midpoint between 4 C and 37 C) and finally a warming study where the cell-matrix constructs were warmed from 4oC to 37oC. A metabolic assay was conducted to investigate the connection between cell attachment and metabolism. The present work allowed us to establish some parameters for cell attachment to the matrix at various temperatures with an epithelial cell line before using expensive stem cells. These data will inform future studies investigating the attachment of induced pluripotent stem cells (ipscs) to decellularized lung matrix at lower temperatures. The second aim of the current study was to create a live lung slice. The live lung slice is a thin (<500μm) viable slice of a human lung that can be maintained in vitro for up to three days. This provides a section of functional lung tissue for future analysis of immune cell response to human lung matrix. Both the creation of the live lung slice as well as the findings from the cell-matrix and attachment studies will direct future steps aimed toward functional lung regeneration, ultimately providing surgeons and patients with better options in lung transplantation. Materials and Methods Preparation of Samples Procurement of Lungs Human lungs and porcine lungs were harvested in similar regions of the lower left lobes. Human lungs rejected for transplantation were procured from the New York Organ Donor Network (NYODN) under a protocol approved by the Institutional Review B oard at Columbia University. Yorkshire pigs (40 50kg) were anesthetized using sodium pentobarbital, mediansternotomy was performed to access the thoracic cavity, and lungs were harvested as per standard protocol. All animal experimental work was performed under a protocol approved by the Columbia University Institutional Animal Care and Use Committee. This process was conducted by people qualified to do so. Decellularization of Human Lung Scaffolds Lungs were stored at -80 C until use. The lungs were then partially thawed and sectioned to 2mm-thick sheets by a deli slicer. Decellularization was done through a series of rinses in solution (as listed below) on an orbital shaker. All slices were initially washed with 2X phosphate buffered saline (PBS) for 15min. The decellularization procedures and solutions are outlined below: (1) SDS: Four 2hr washes with 1.8mM sodium dodecyl sulfate (SDS) each followed by a 5 minute wash with deionized water (dh 2O) and a 15 minute 2x PBS wash. (2) CHAPS: Four 2hr washes with 8mM 3-[(3- Cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS). This was followed by a 5min wash with dh 2O and a 15min 2X PBS (3) 3-Step method: One 2hr wash with 3% Tween-20, one 2hr wash with 4% sodium deoxycholate, and lastly, one 1hr wash with 0.1% peracetic acid. All slices were then subjected to alternating 1X PBS and dh 2O washes (two of each total). Spring 2014 Volume 8 12

4 Maintenance of Small Airway Epithelial Cells (SAECs) Cells were received from ATCC and Lonza companies and retrieved from storage in liquid nitrogen. Cells were thawed at 37 o C through a series of sterile 1x PBS washes and trypsin. Media was changed every 2 to 3 days after a 1x PBS wash in order. Cells were not trypsinized until 75% confluency was observed. SAECs were maintained in small airway epithelial growth medium. Small Airway Epithelial Growth Media consists of bovine pituary extract, epidermal growth factor, hydrocortisone, epinephrine, triiodo- L-thyronine, transferrin, holo, retonoic acid, 1% pen/strep and bovine serum albumin (fatty acid free) as per the Lonza protocol. DMEM consisted of DMEM and 1% pen/strep. ECM Punches After decellularization, 7mm-diameter discs of decellularized tissue were punched using a biopsy punch under sterile conditions and used in experiments. Decellularized matrices were then attached to 96-well plates using fibrin glue, created using a 1:3 ratio of fibrinogen to thrombin. Cell Attachment Studies Temperature Coating Study Cell attachment was initially studied using a coating experiment. In this study, tissue culture plastic was coated with 0.1% gelatin, 0.1% collagen I, and 0.1% decellularized digested ECM matrix, and a decellularized matrix punch. Controls included an uncoated tissue culture plastic well and a well filled with plain media, as shown below. Cells were seeded at a density of 20,000 cells per well and allowed to incubate at 37 o C (temperature at which cells thrive), 4 o C (temperature at which organs are stored during transplantation), and 20 C (room temperature). Multiple trials were conducted for each temperature. Metabolic assays, Live/Dead staining, and CFSE staining were conducted at different time intervals to quantify cell attachment, morphology, and viability. Assays were performed at 6 hours, 24 hours, 48 hours, and 72 hours after cell seeding. Figure 2: Coating Study Experimental Set-Up. Physical Attachment Assay In order to test the mechanical strength of cell attachment at 48 hours the coated and scaffold filled 96-well plates were placed right side up in a box of buffered saline until all the wells were filled with water. The plates were then turned upside down in order to allow any unattached cells to wash away. This was done in order to assess the strength of the cell attachment. Immunofluorescent images were taken in order to quantify cell attachment. Warming Study After 48 hours, samples stored at 4 o C were allowed to incubate at 37 o C for an additional 24 hours to see if cells attached and were viable after a temperature shock. To assess this fluorescent staining images were taken and a metabolic assay was conducted. Metabolic Assay Metabolic activity of the cells was measured using Alamar Blue reagent according to the manufacturer s instructions. The reagent was added 24 hours before respective time limit and incubated under standard culture conditions. Samples were analyzed using fluorescence spectroscopy and absorbance was measured at 540nm with reference wavelength of 570nm. Fluorescent Staining Samples were stained using carboxyfluorescein succinimidyl ester (CFSE) and Spring 2014 Volume 8 13

5 calcein AM to confirm live cells in the culture, while ethidium bromide staining was used to identify the dead cells. CFSE stain was prepared at a working concentration of 5μM in 1X PBS and the live/dead stain was prepared by combining 20μL of ethidium bromide and 5μL of calcein AM in 10mL of 1X PBS. Samples were rinsed with 1X PBS and then allowed to incubate at room temperature with the respective stain. After aspirating the stain, samples were washed three to four times with 1X PBS and imaged. Live Lung Slice Porcine Lung Thawed porcine lung airways were perfused with gelatin concentrations of 1%, 10%, 15%, and 20% Type A Gelatin solutions using a catheter and tubing in order to create a stiffer tissue to be cut for the live lung slice. Live lung slices were cut using a scalpel for thicker pieces and a vibratome for thinner slices. Gelatin was either heated away or cut off after slice was made. DAPI was used to visualize cell nuclei, and images were taken using a fluorescent microscope. Results Cell Attachment Studies Coating Study Immunofluorescent Images All images shown are of tissue culture plastic (TCP), gelatin, and digested decellularized extracellular matrix (ECM). TCP was a non-coated positive cell attachment control, gelatin as a supportive coating substance, and digested ECM as a chemical representation of decellularized ECM. CFSE staining showed similar results to the live/dead staining. Therefore, CFSE data are not shown. Live cells are stained green, and dead cells stained red. All images obtained with 10X objective. 6 hour images are the control for this study, to show minimum amount of attachment and cell growth. Tissue Culture Plastic 6 hours Figure 3: All images taken at 6 hours (A) Brightfield Image of TCP at 4C (B) Brightfield Image of TCP at 20C (C) Brightfield Image of TCP at 37C (D) Fluorescent LIve/Dead Stain of TCP at 4C (E) Fluorescent Live/Dead Stain of TCP at 20C (F) Fluorescent Live/Dead Stain of TCP at 37C. 24 hours Figure 4: All images taken at 24 hours (A) Brightfield Image of TCP at 4C (B) Brightfield Image of TCP at 20C (C) Brightfield Image of TCP at 37C (D) Fluorescent LIve/Dead Stain of TCP at 4C (E) Fluorescent Live/Dead Stain of TCP at 20C (F) Fluorescent Live/Dead Stain of TCP at 37C. Spring 2014 Volume 8 14

6 Gelatin 48 hours 6 hours Figure 5: All images taken at 48 hours (A) Brightfield Image of TCP at 4C (B) Brightfield Image of TCP at 20C (C) Brightfield Image of TCP at 37C (D) Fluorescent LIve/Dead Stain of TCP at 4C (E) Fluorescent Live/Dead Stain of TCP at 20C (F) Fluorescent Live/Dead Stain of TCP at 37C. 72 hours Figure 7: All images taken at 6 hours (A) Brightfield Image of Gelatin at 4C (B) Brightfield Image of Gelatin at 20C (C) Brightfield Image of Gelatin at 37C (D) Fluorescent LIve/Dead Stain of Gelatin at 4C (E) Fluorescent Live/Dead Stain of Gelatin at 20C (F) Fluorescent Live/Dead Stain of Gelatin at 37C. 24 hours Figure 6: All images taken at 72 hours (A) Brightfield Image of TCP at 4C (B) Brightfield Image of TCP at 20C (C) Brightfield Image of TCP at 37C (D) Fluorescent LIve/Dead Stain of TCP at 4C (E) Fluorescent Live/Dead Stain of TCP at 20C (F) Fluorescent Live/Dead Stain of TCP at 37C. Figure 8: All images taken at 24 hours (A) Brightfield Image of Gelatin at 4C (B) Brightfield Image of Gelatin at 20C (C) Brightfield Image of Gelatin at 37C (D) Fluorescent LIve/Dead Stain of Gelatin at 4C (E) Fluorescent Live/Dead Stain of Gelatin at 20C (F) Fluorescent Live/Dead Stain of Gelatin at 37C. Spring 2014 Volume 8 15

7 ECM Matrix 48 hours 6 hours Figure 9: All images taken at 48 hours (A) Brightfield Image of Gelatin at 4C (B) Brightfield Image of Gelatin at 20C (C) Brightfield Image of Gelatin at 37C (D) Fluorescent LIve/Dead Stain of Gelatin at 4C (E) Fluorescent Live/Dead Stain of Gelatin at 20C (F) Fluorescent Live/Dead Stain of Gelatin at 37C. 72 hours Figure 11: All images taken at 6 hours (A) Brightfield Image of digested ECM at 4C (B) Brightfield Image of digested ECM at 20C (C) Brightfield Image of digested ECM at 37C (D) Fluorescent LIve/Dead Stain of digested ECM at 4C (E) Fluorescent Live/Dead Stain of digested ECM at 20C (F) Fluorescent Live/Dead Stain of digested ECM at 37C. 24 hours Figure 10: All images taken at 72 hours (A) Brightfield Image of Gelatin at 4C (B) Brightfield Image of Gelatin at 20C (C) Brightfield Image of Gelatin at 37C (D) Fluorescent LIve/Dead Stain of Gelatin at 4C (E) Fluorescent Live/Dead Stain of Gelatin at 20C (F) Fluorescent Live/Dead Stain of Gelatin at 37C. Figure 12: All images taken at 24 hours (A) Brightfield Image of digested ECM at 4C (B) Brightfield Image of digested ECM at 20C (C) Brightfield Image of digested ECM at 37C (D) Fluorescent LIve/Dead Stain of digested ECM at 4C (E) Fluorescent Live/Dead Stain of digested ECM at 20C (F) Fluorescent Live/Dead Stain of digested ECM at 37C. Spring 2014 Volume 8 16

8 48 hours Figure 13: All images taken at 48 hours (A) Brightfield Image of digested ECM at 4C (B) Brightfield Image of digested ECM at 20C (C) Brightfield Image of digested ECM at 37C (D) Fluorescent LIve/Dead Stain of digested ECM at 4C (E) Fluorescent Live/Dead Stain of digested ECM at 20C (F) Fluorescent Live/Dead Stain of digested ECM at 37C. 72 hours addition, most of the cells stained seem to still be alive, as indicated by the majority of green colored cells. After 24 hours, cells at 4 C are slightly more attached, exhibiting a more uniform pattern. They are still, however, more rounded, indicating a lesser degree of attachment in comparison to cells at the other two temperatures. There is a higher density of cells at 20 C with a more flattened shape. At 37 C cells are both uniform and flattened and showing a greater level of confluency and higher density of cells. This pattern continues on to 48 hours, as the cells at 4 C are still rounded. The cells at 20 C are showing a similar morphology of the cells at 37 C: more flattened and confluent. By 72 hours, it seems as though the cells at maximum confluency at 37 C and with a medium amount of confluency and flattened morphology at 20 C. At 4 C, cells seem to start to exhibit the beginning stages of flattened morphology, but with a sparse cell density. Metabolic Assays Figure 14: All images taken at 72 hours (A) Brightfield Image of digested ECM at 4C (B) Brightfield Image of digested ECM at 20C (C) Brightfield Image of digested ECM at 37C (D) Fluorescent LIve/Dead Stain of digested ECM at 4C (E) Fluorescent Live/Dead Stain of digested ECM at 20C (F) Fluorescent Live/Dead Stain of digested ECM at 37C. All three coating types exhibited similar results in terms of cell morphology with gelatin maintaining the greatest promotion of cellular attachment. At 6 hours, the cells are 4 C, the cells exhibit a rounded shape and seem to attach to the side of the wells after 3 rinses with 1X PBS. At 20 C, the cells exhibit a more uniform pattern and have started to flatten out, while at 37 C the cells are both attached, uniform, and flattened out. In Spring 2014 Volume 8 17

9 Fluorescent Image of Digested ECM 4C (E) Fluorescent Image of Digested ECM 20C (F) Fluorescent Image of Digested ECM 37C. Figure 15: Cell Metabolic Assays (A) between 6 and 24 hours (B) between 24 and 48 hours (C) between 48 and 72 hours. Between 6 hours and 24 hours, cells on gelatin had the most metabolic activity at 37 C while cells on digested ECM had the most metabolic activity at 4 C. Overall, cells at 37 C had the about two times the amount of metabolic activity in comparison to cells at 4 C. A similar pattern was observed for the metabolic activity between 24 and 48 hours, however the emission was about 10,000 nm greater. Finally between 48 and 72 hours its seems as though the cells at 37 C had a consistent metabolic pattern. In addition, the gap between cell metabolism between cells at 4 C and 37 C became smaller indicating that after 48 hours, the cells at 4 C began to metabolize more than they had before. Three trials were conducted, however due to a mishap in one of the trials only two contributed to the data set. Since n=2 statistical analysis was not possible. Physical Attachment Assay Fluorescent Images The physical attachment study was conducted in order to test the degree of cell attachment. All images shown above are for digested ECM Matrix, since the chemical coating makeup is most similar to our matrix type. As shown by Figure 16 A and D it seems that there was a very weak degree of cell attachment at 4 C, whereas at 20 C and 37 C most of the cells that had been attached originally at 48 hours (Figure 13 C and F) remained for the most part attached to the coated surface. It should also be noted that during the physical attachment study at 20 C there were a number of cells that died during the procedure. Warming Study Figure 17: Fluorescent Images of Warming Study. Cells were incubated at 37C for 24 hours after incubation at 4C for 48 hours for heat shock analysis (A) Brightfield Image of TCP (B) Brightfield Image of Gelatin (C) Brightfield Image of digested ECM (D) Fluorescent Image of TCP (E) Fluorescent Image of Gelatin (F) Fluorescent Image of digested ECM. Figure 16: Images taken after submersion in buffered saline as previously described (A) Brightfield Image of Digested ECM 4C (B) Brightfield Image of Digested ECM 20C (C) Brightfield Image of Digested ECM 37C (D) Fluorescent Images indicate that cells do not suffer from immense heat shock after cells are incubated at 37 C for 24 hours after being incubated at 4 C for 48 hours. This is supported by the fact that there are live cells on all three surface types as indicate by the green stain. In addition, it seems that the cells are beginning to become confluent an d are having the flattened morphology that was seen at 20 C and 37 C on Spring 2014 Volume 8 18

10 well-attached cells. Gelatin seems to be the most effective coating surface in terms of cell retention and attachment as indicated by the greater number of cells. This means that the cells are capable of surviving two very different temperatures if need be. Live Lung Slice Figure 19: Lung slice sectioned by hand with 20% Type A gelatin perfusion (left). Lung slice sectioned by vibratome (right). In order to test the efficacy of the live lung slice, the porcine lungs were perfused with different concentrations of Type A gelatin in order to allow the slice to retain its original structure. 10% gelatin concentration in 1X PBS proved to be most effective. DAPI staining was used to visualize the clearance of gelatin from the airways and vessels, which can be clearly seen. Figure 18: Cannulation and Perfusion set-up for injection of gelatin into lung airways and vessels for live lung slice. Figure 19: DAPI blue stain of cell nuclei to visualize lung airway and vessel. Discussion This study was conducted in order to analyze cell attachment at various temperatures. This was done in order to determine the optimal temperature and time needed for the recellularization portion of lung reconditioning and regeneration for more effective organ transplantation. This was first studied through the "coating study" where tissue culture plastic was coated with gelatin, or digested decellularized lung Spring 2014 Volume 8 19

11 extracellular matrix. Although all surfaces were successful in promoting some degree of cell attachment, the most effective coating was gelatin, i.e. gelatin retained the greatest number of attached cells. This indicates the possible use of coating lung matrix with gelatin during the recellularization process. Immunofluorescent staining indicated that there was cell attachment at all three temperatures tested: 4 C, 20 C, and 37 C. It was initially expected that at 4 C there would be no attachment, but the coating study indicated otherwise. The live/dead stain indicated that most of the cells that remained attached were still alive in contrast those that died or failed to attach. The CFSE stain reinforced this finding, showing similar densities and number of live cells as well for each respective set-up. There seemed to be a small degree of cell attachment at 4 C, which was initially not expected. This was further supported by the physical attachment assay, in which the cells remained attached even after attempted physical de-attachment by buffered saline. For the first 48 hours, the cells at 4 C had a rounded morphology, indicating that while the cells were attached they were not completely attached to the surface. This is further supported by the metabolic studies in which cells at 4 C seemed to not metabolize much more than the negative control which contained no cells. This indicates that while the cells were alive, and slightly attached to the surface that they were metabolizing enough to survive. This information is useful to know, because most transplanted organs are left on ice between procurement from the donor and implant into the recipient. Therefore, by mimicking these conditions, we can determine the optimal temperature at which these cells can attach and survive. The 37 C cells seemed to be the most effective with a high density of surviving cells and with consistent flattened confluent appearance as early as 6 hours. In addition, the metabolic assay indicated a high amount of metabolic activity in comparison to the other two temperatures and the control. The problem, however, is that it is not possible to keep a transplant organ at 37 C for too long due to the difficulty of maintaining the vasculature. Therefore, the 20 C results seemed to be the most promising. A high number of cells seemed to attach to the surfaces in comparison to the 4 C. By 24 hours, the cells began to flatten out and by 48 hours a high level of confluency was observed, meaning that the cells were attached, living and behaving in a similar fashion to cells kept at 37 C, which is ideal. In addition, there seemed to be a high level of metabolism from the cells at 20 C, meaning that they were functioning properly and attaching to the surface and were able to survive at that temperature. The next steps in a future study would be to see if simultaneous decellularization and vasculature preservation of the lungs could be possible at a temperature between 4 C and 20 C. Other future work includes co-culture studies with epithelial cells and fibroblasts or mesenchymal stem cells (MSCs), to see if there is a greater degree of attachment and metabolism occurring with the mixture of cells and cell secretions. Fibroblasts are an important support cell in the airways. Both fibroblasts and MSCs are known to secrete helpful proteins and substances into the cellular environment to aid in attachment and cell viability. Ultimately, these experiments will be done using ips cells, with potential co-culture, in order determine the most effective recellularization method. The second study included the production of a thin live lung slice. It was determined that using 12% gelatin to perfuse the lung airways was the most effective Type A Gelatin concentration. Once the gelatin hardened, the lung was sliced effectively using either a scalpel or vibratome. The DAPI staining indicated clearance of the airways and the vessels of solid gelatin. Problems encountered with gelatin perfusion included the stiffness of the lung after gelatin injection and the gelatinization of the liquid gelatin before perfusion was completed. The value of a thin live lung slice lies in future investigation of vessels and airways contractility with the addition of acetylcholine. A key future application of the live lung slice includes adding a section of decellularized matrix into a native live lung slice to observe their effects on immune cells for future immunological studies. These studies provide the basis for establishing decellularization/recellularization conditions for reconditioning lungs under the appropriate temperature as well as establishing a method for generating a live lung slice model for future studies geared toward understanding immune response in a bioengineered chimeric lung. Spring 2014 Volume 8 20

12 References 1. Badylak, S.F., et al. (2009). Extracellular matrix as a biological scaffold material: Structure and Function, ActaBiomaterialia 5: Badylak, S.F. (2002). The extracellular matrix as a scaffold for tissue reconstruction, Cell and Developmental Biology 13: Badylak, S.F. et al. (2011). Whole-Organ Tissue Engineering: Decellularization and Recellularization of Three-Dimensional Matrix Scaffolds, Annu. Rev. of Biomed. Eng. 13: Boyle, J., Buckley, R. (2007). Population Prevalence of Diagnosed Primary Immunodeficiency Diseases in the United States, Journal of Clinical Immunology 27: Brown, B.N. et al. (2009). Surface characterization of extracellular matrix scaffolds, Biomaterials 31: Freytes, D.O. et al. (2008) Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix, Biomaterials 29: Ott, H.C., et al. (2010). Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med 16(8): p Petersen, T.H., et al., Matrix Composition and Mechanics of Decellularized Lung Scaffolds. Cells, tissues, organs, Reing, J.E. et al. (2009). Degradation products of extracellular matrix affect cell migration and proliferation, Tissue Engineering: Part A15: Sykes M., Nikolic B. (2005). Treatment of severe autoimmune disease by stem-cell transplantation. Nature 435: Takahashi, K. "Induction of pluripotent stem cells from fibroblast cultures." Nature Protocols (2007): Print. < n12/pdf/nprot pdf>. Spring 2014 Volume 8 21