- Email: [email protected]
A Perfusion Bioreactor for Intestinal Tissue Engineering
Journal of Surgical Research 142, 327–331 (2007) doi:10.1016/j.jss.2007.03.039
A Perfusion Bioreactor for Intestinal Tissue Engineering Stephen S. Kim, M.D.,*,†,‡,§,1 Rebecca Penkala, B.S.,‡ and Parwiz Abrahimi,‡ *Seattle Children’s Hospital Research Institute, Children’s Hospital and Regional Medical Center; †Department of Surgery, University of Washington School of Medicine; ‡Department of Bioengineering, University of Washington; and §Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington Submitted for publication January 9, 2007
INTRODUCTION Background. Short gut syndrome is a devastating clinical problem with limited long-term treatment options. A unique characteristic of the normal intestinal epithelium is its capacity for regeneration and adaptation. Despite this tremendous capacity in vivo, one of the major limitations in advancing the understanding of intestinal epithelial differentiation and proliferation has been the difficulty in maintaining primary cultures of normal gut epithelium in vitro. A perfusion bioreactor system has been shown to be beneficial in long-term culture and bioengineering of a variety of tissues. The purpose of this study is to design and fabricate a perfusion bioreactor for intestinal tissue engineering. Materials and methods. A perfusion bioreactor is fabricated using specific parameters. Intestinal epithelial organoid units harvested from neonatal rats are seeded onto biodegradable polymer scaffolds and cultured for 2 d in the bioreactor. Cell attachment, viability, and survival are assessed using MTT assay, scanning electron micrograph, and histology. Results. A functional perfusion bioreactor was successfully designed and manufactured. MTT assay and scanning electron micrograph demonstrated successful attachment of viable cells onto the polymer scaffolds. Histology confirmed the survival of intestinal epithelial cells seeded on the scaffolds and cultured in the perfusion bioreactor for 2 days. Conclusions. A functional perfusion bioreactor can be successfully fabricated for the in-vitro cultivation of intestinal epithelial cells. With further optimization, the perfusion bioreactor may be a useful in in-vitro system for engineering new intestinal tissue. © 2007 Elsevier Inc. All rights reserved.
Key Words: regeneration; intestinal tissue engineering; bioreactor; short gut syndrome.
Short gut syndrome is a form of intestinal failure, which results from the loss of more than two thirds of the normal jejunal-ileal length. This clinical condition afflicts both adults and children and is characterized by diarrhea, dehydration, malabsorption, and progressive malnutrition. The mainstay of therapy is the use of parenteral nutrition (PN). Although life-saving, PN is extremely expensive and associated with significant morbidity and mortality. Using the principles of tissue engineering, our laboratory has been investigating the fabrication of functional intestinal tissue using isolated cells seeded on synthetic biodegradable polymer scaffolds. Previous studies in small animal models have demonstrated the engraftment and survival of intestinal cells transplanted on tubular polymer scaffolds with regeneration of new tissue with a neomucosa and tissue morphology resembling small intestine [1– 4]. One of the unique characteristics of the normal intestinal epithelium is its capacity for renewal and adaptation. Both of these processes are thought to involve intestinal stem cells residing within the crypts of the mucosa. Despite this tremendous capacity in vivo, one of the major limitations in advancing the understanding of intestinal epithelial proliferation and differentiation has been the difficulty in maintaining primary cultures of normal gut epithelium. A perfusion bioreactor system has been shown to be beneficial in the long-term culture and bioengineering of a variety of tissues [5–9]. A bioreactor may provide an in vitro environment that more closely mimics the normal physiological conditions. The purpose of this study was to design and fabricate a perfusion bioreactor for intestinal tissue engineering. MATERIALS AND METHODS
1
To whom correspondence and reprint requests should be addressed at Seattle Children’s Research Institute, Children’s Hospital and Regional Medical Center, 4800 Sand Point Way, NE, W7729, Seattle, WA 98105. E-mail: [email protected].
Animals Nonfasted 7-d old neonatal Lewis rats (Charles River Laboratories, Wilmington, MA) were used as intestine donors for isolation of
327
0022-4804/07 $32.00 © 2007 Elsevier Inc. All rights reserved.
328
JOURNAL OF SURGICAL RESEARCH: VOL. 142, NO. 2, OCTOBER 2007
intestinal epithelial organoid units. Animals were housed in the Animal Research Facility at the Seattle Children’s Research Institute, Seattle, Washington, in accordance with National Institute of Health guidelines for the care of laboratory animals. Animals were maintained in a temperature-regulated environment (24°C) on a 12-h light-dark cycle, housed in cages with soft bedding and microisolator cover.
Polymer Scaffold Fabrication Microporous 3-D synthetic biodegradable polymer tubes were fabricated from nonwoven sheets of polyglycolic acid (PGA) fibers (fiber diameter 15 m; mesh thickness 2 mm; bulk density 60 mg/cm 3; porosity ⬎96%; mean pore size 250 m; Albany International, Albany, NY) as previously described [10]. The tubes were sprayed on the outer surface with a solution of polylactic acid (PLLA, Sigma) (5% wt/vol). The polymer tubes were sterilized with ethylene oxide, then coated with collagen Type 1 (Vitrogen; Cohesion Technologies, Palo Alto, CA). The scaffolds were rinsed with phosphate buffered saline (PBS) and Hanks balanced salt solution (HBSS) prior to cell seeding.
Perfusion Bioreactor Design and Fabrication The design for the perfusion bioreactor was adapted from the previously reported system [6]. The bioreactor consisted of a multichannel peristaltic pump, culture medium reservoir, oxygenation and gas exchange unit, air trap, and cell-polymer construct housing unit (Fig. 1). The culture medium was pumped at a flow rate of 1.5 mL/min from a 100 mL reservoir through the oxygenation and cellpolymer housing units and recirculated back to the reservoir. The adequacy of the oxygenation and gas exchange unit was assessed by measuring pH, PO 2, and PCO 2 of the culture medium with varying incubator CO 2 concentrations using a Bioprofile 400 Analyzer (NovaBiomed, Waltham, MA). The culture medium analysis was performed in duplicate (CO 2 10% and 15%) or triplicate (CO 2 5%) studies. The bioreactor circuit was modified using three-way stopcocks to allow the addition of a dynamic cell-seeding loop in continuity with the culture loop as well as sampling sites for culture medium analysis. The culture medium was collected distal to the cell-polymer housing unit for analysis. The entire circuit was sterilized by autoclave or ethylene oxide gas and maintained at 37°C with 8% CO 2 supplementation during cell seeding and culture. The bioreactor circuit was primed initially with PBS then with culture medium prior to loading with the cell-polymer constructs.
Cell Isolation Intestinal epithelial organoid units were harvested from nonfasted neonatal Lewis rats using an enzymatic dissociation technique as described by Evans [11]. Briefly, under isoflurane inhalational anesthesia, the entire length of small intestine was harvested, stripped of its mesentery, and placed in HBSS on ice. The intestines were flushed with cold HBSS, split open and cut into 2- to 3-mm fragments. The intestinal fragments were further washed with HBSS, sharply minced into ⬍1 mm 3 pieces, and then enzymatically dissociated (0.1 mg/mL dispase, neutral protease Type 1; Roche Applied Science, and 300 U/mL collagenase type 2, (Worthington) at room temperature on an orbital shaking platform at 80 rpm for 25 min. After mechanical agitation, the intestinal epithelial organoid units were further purified by centrifugation in a solution of Dulbecco’s modified Eagle’s medium, 2.5% heat-inactivated fetal calf serum, and 2% sorbitol (Sigma Chemical Co., St. Louis, MO) at 300 rpm for 2 min. The resulting pellet was resuspended in culture medium and counted.
Perfusion Bioreactor Culture The tubular polymer scaffolds were placed into the cell-polymer housing unit under sterile conditions after the circuit had been primed with PBS and culture medium. The intestinal epithelial organoid units were resuspended in culture medium and dynamically seeded through the seeding loop of the bioreactor circuit as previously described [6]. After the seeding period, the three-way stopcocks were turned to exclude the seeding loop, and the reservoir bottle containing the cell suspension was replaced with fresh warmed culture medium. The cell-polymer constructs (n ⫽ 4) were cultured under flow conditions for 2 d. Prior to harvest, the cells were incubated in 1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO) solution for an additional 12 h to assess general metabolic activity and distribution of cells within the scaffolds. The constructs were then harvested and processed for scanning electron microscopy and histology.
RESULTS Polymer Scaffold Fabrication
Highly porous tubular 3-D polymer scaffolds were fabricated with dimensions of 5 mm o.d., 2 mm i.d., and 10 mm length (Fig. 2). The devices were ⬎95% porous with a mean pore size approximately 250 m. The outer coating of PLLA provided structural integrity to the polymer tubes as previously reported [10]. Perfusion Bioreactor Design and Fabrication
A functional perfusion bioreactor culture system was successfully designed and manufactured (Fig. 3). Analysis of the culture medium under flow conditions demonstrated adequate oxygenation of the medium within the circuit. There was good correlation between culture medium pH and PCO 2 levels with changes in incubator CO 2 concentration demonstrating proper function of the gas exchange unit (Fig. 4). Intestinal Epithelial Organoid Unit Isolation FIG 1. Schematic diagram of the perfusion bioreactor. Dashed arrow depicts the seeding loop. Solid arrows depict the bioreactor loop.
Approximately 1.4 to 1.7 ⫻ 10 5 organoid units were harvested from each 7-d old neonatal Lewis rat. The organoid units were resuspended in culture medium at
KIM, PENKALA, AND ABRAHIMI: INTESTINAL TISSUE ENGINEERING
329
DISCUSSION
FIG 2. Highly porous biodegradable polymer tube made from polyglycolic acid fiber mesh. (A) Photomicrograph. (B) Low magnification scanning electron micrograph.
Short gut syndrome is a form of intestinal failure that has limited long-term treatment options. Tissue engineering approaches have been investigated as an alternative approach to treatment of intestinal failure and other forms of end stage organ disease. Our laboratory has been investigating the fabrication of functional living intestinal tissue using isolated cells combined with highly porous biodegradable polymer matrices. Previous studies in small animals have demonstrated the engraftment and survival of isolated intestinal epithelial organoid units transplanted on biodegradable polymer tubes, with regeneration of new tissue that morphologically resembled small intestine [1– 4]. Because of the difficulty in maintaining primary cultures of normal intestinal epithelium, researchers have developed methods of isolating intestinal epithelial organoid units, which preserve epithelial-mesenchymal interactions critical for epithelial proliferation and differentiation. Epithelial organoid units are also thought to contain intestinal stem cells and thus possess the capacity for regenerating new intestinal tissue. To rigorously investigate the mechanisms involved in intestinal epithelial proliferation, differentiation, and regeneration, we have hypothesized that a perfusion bioreactor system would be advantageous for in vitro culture and conditioning of intestinal cells as has been shown for other tissues [6 –9]. An in vitro system would allow for more uniform and reliable control of the environment in which the cells are initially exposed. It would minimize the period of hypoxia, which would potentially enhance cellular survival, proliferation, and differentiation. The effects of various trophic factors may be investigated in a more direct and precise manner than may be possible in an in vivo model. In addition, many investigators have demonstrated the beneficial effects of dynamic seeding tech-
a density of 2.5 ⫻ 10 4 organoid units/mL. Forty milliliters of the suspension was placed in the reservoir bottle and dynamically seeded through the seeding loop of the perfusion bioreactor. Perfusion Bioreactor Culture
After dynamic seeding and 2 d in flow culture, scanning electron micrograph demonstrated the successful attachment of intestinal epithelial organoid units onto the PGA polymer scaffold (Fig. 5). MTT assay demonstrated uniform distribution of viable cells throughout the polymer scaffold (Fig. 6). Histology confirmed the presence of viable clusters of cells with associated extracellular matrix within the PGA polymer scaffolds (Fig. 7). The cells from the organoid units appear to have coalesced into larger clusters and spread along the fibers of the scaffold, potentially as the initial steps in reorganization.
FIG 3.
Perfusion bioreactor culture system.
330
JOURNAL OF SURGICAL RESEARCH: VOL. 142, NO. 2, OCTOBER 2007
FIG 4. Culture medium analysis. Diamond: 5% CO 2 concentration; square: 10% CO 2 concentration; circle: 15% CO 2 concentration. (A) Culture medium PO 2 levels under varying incubator CO 2 concentrations. (B) Culture medium PCO 2 levels under varying incubator CO 2 concentrations. (C) Culture medium pH levels under varying incubator CO 2 concentrations.
niques in regards to cell attachment and uniform cell distribution within the polymer matrices [6, 12, 13]. In this study, we have been able to design and manufacture a functional perfusion bioreactor system for intestinal tissue engineering. This system can be sterilized and maintained under controlled conditions in vitro. It provides adequate oxygenation and CO 2 gas exchange. We have demonstrated that intestinal epi-
thelial organoid units can be dynamically seeded onto tubular polymer scaffolds and survive under flow conditions for 2 d. Further studies will be needed to optimize the parameters for dynamic seeding, optimize the polymer scaffold to enhance cellular attachment, investigate the long-term in vitro cultivation of intestinal cells seeded on biodegradable polymer scaffolds, and delineate the effects trophic factors for new tissue development. Continued efforts into long-term in vitro culture of intestinal epithelial and stem cells under
FIG 5. Scanning electron micrograph demonstrating successful attachment of intestinal epithelial cells onto the biodegradable polymer scaffold after dynamic seeding and 2 d of flow culture.
FIG 6. MTT assay demonstrating the uniform distribution of viable cells on the polymer scaffold after seeding and 2 d of flow culture.
KIM, PENKALA, AND ABRAHIMI: INTESTINAL TISSUE ENGINEERING
331
gradable polymer scaffolds for tissue engineering of small intestine. Transplantation 1999;67:227.
FIG 7. Hematoxylin and eosin histology demonstrating the presence of viable cells within the polymer scaffold 2 d after dynamic seeding and flow culture (magnification ⫻40).
flow conditions may yield important insights into the mechanisms involved in tissue morphogenesis and regeneration, and ultimately lead to the development of functional replacement tissues for the treatment of short gut syndrome. ACKNOWLEDGMENTS This work was funded by generous grants from the Department of Surgery, Children’s Hospital and Regional Medical Center and University of Washington, and the Seattle Children’s Hospital Steering Committee Award.
REFERENCES 1.
Kim SS, Kaihara S, Benvenuto MS, et al. Regenerative signals for intestinal epithelial organoid units transplanted on biode-
2.
Kaihara S, Kim SS, Benvenuto MS, et al. Successful anastomosis between tissue-engineered intestine and native small bowel. Transplantation 1999;67:241.
3.
Kim SS, Kaihara S, Benvenuto MS, et al. Effects of anastomosis of tissue-engineered neo-intestine to native small bowel. J Surg Res 1999;87:6.
4.
Kaihara S, Kim SS, Kim BS, et al. Long-term follow-up of tissue-engineered intestine after anastomosis to native small bowel. Transplantation 2000;69:1927.
5.
Kim SS, Utsunomiya H, Koski JA, et al. Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels. Ann Surg 1998;228:8.
6.
Kim SS, Sundback CA, Kaihara S, et al. Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system. Tissue Eng 2000;6:39.
7.
Portner R, Nagel-Heyer S, Goepfert C, et al. Bioreactor design for tissue engineering. J Biosci Bioeng 2005;100:235.
8.
Abousleiman RI, Sikavitsas VI. Bioreactors for tissues of the musculoskeletal system. Adv Exp Med Biol 2006;585:243.
9.
Holtorf HL, Jansen JA, Mikos AG. Modulation of cell differentiation in bone tissue engineering constructs cultured in a bioreactor. Adv Exp Med Biol 2006;585:225.
10.
Mooney DJ, Mazzoni CL, Breuer C, et al. Stabilized polyglycolic acid fibre-based tubes for tissue engineering. Biomaterials 1996;17:115.
11.
Evans GS, Flint N, Somers AS, et al. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J Cell Sci 1992;101:219.
12.
Vunjak-Novakovic G, Obradovic B, Martin I, et al. Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog 1998;14:193.
13.
Kim BS, Putnam AJ, Kulik TJ, et al. Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng 1998;57:46.
A Perfusion Bioreactor for Intestinal Tissue Engineering Stephen S. Kim, M.D.,*,†,‡,§,1 Rebecca Penkala, B.S.,‡ and Parwiz Abrahimi,‡ *Seattle Children’s Hospital Research Institute, Children’s Hospital and Regional Medical Center; †Department of Surgery, University of Washington School of Medicine; ‡Department of Bioengineering, University of Washington; and §Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington Submitted for publication January 9, 2007
INTRODUCTION Background. Short gut syndrome is a devastating clinical problem with limited long-term treatment options. A unique characteristic of the normal intestinal epithelium is its capacity for regeneration and adaptation. Despite this tremendous capacity in vivo, one of the major limitations in advancing the understanding of intestinal epithelial differentiation and proliferation has been the difficulty in maintaining primary cultures of normal gut epithelium in vitro. A perfusion bioreactor system has been shown to be beneficial in long-term culture and bioengineering of a variety of tissues. The purpose of this study is to design and fabricate a perfusion bioreactor for intestinal tissue engineering. Materials and methods. A perfusion bioreactor is fabricated using specific parameters. Intestinal epithelial organoid units harvested from neonatal rats are seeded onto biodegradable polymer scaffolds and cultured for 2 d in the bioreactor. Cell attachment, viability, and survival are assessed using MTT assay, scanning electron micrograph, and histology. Results. A functional perfusion bioreactor was successfully designed and manufactured. MTT assay and scanning electron micrograph demonstrated successful attachment of viable cells onto the polymer scaffolds. Histology confirmed the survival of intestinal epithelial cells seeded on the scaffolds and cultured in the perfusion bioreactor for 2 days. Conclusions. A functional perfusion bioreactor can be successfully fabricated for the in-vitro cultivation of intestinal epithelial cells. With further optimization, the perfusion bioreactor may be a useful in in-vitro system for engineering new intestinal tissue. © 2007 Elsevier Inc. All rights reserved.
Key Words: regeneration; intestinal tissue engineering; bioreactor; short gut syndrome.
Short gut syndrome is a form of intestinal failure, which results from the loss of more than two thirds of the normal jejunal-ileal length. This clinical condition afflicts both adults and children and is characterized by diarrhea, dehydration, malabsorption, and progressive malnutrition. The mainstay of therapy is the use of parenteral nutrition (PN). Although life-saving, PN is extremely expensive and associated with significant morbidity and mortality. Using the principles of tissue engineering, our laboratory has been investigating the fabrication of functional intestinal tissue using isolated cells seeded on synthetic biodegradable polymer scaffolds. Previous studies in small animal models have demonstrated the engraftment and survival of intestinal cells transplanted on tubular polymer scaffolds with regeneration of new tissue with a neomucosa and tissue morphology resembling small intestine [1– 4]. One of the unique characteristics of the normal intestinal epithelium is its capacity for renewal and adaptation. Both of these processes are thought to involve intestinal stem cells residing within the crypts of the mucosa. Despite this tremendous capacity in vivo, one of the major limitations in advancing the understanding of intestinal epithelial proliferation and differentiation has been the difficulty in maintaining primary cultures of normal gut epithelium. A perfusion bioreactor system has been shown to be beneficial in the long-term culture and bioengineering of a variety of tissues [5–9]. A bioreactor may provide an in vitro environment that more closely mimics the normal physiological conditions. The purpose of this study was to design and fabricate a perfusion bioreactor for intestinal tissue engineering. MATERIALS AND METHODS
1
To whom correspondence and reprint requests should be addressed at Seattle Children’s Research Institute, Children’s Hospital and Regional Medical Center, 4800 Sand Point Way, NE, W7729, Seattle, WA 98105. E-mail: [email protected].
Animals Nonfasted 7-d old neonatal Lewis rats (Charles River Laboratories, Wilmington, MA) were used as intestine donors for isolation of
327
0022-4804/07 $32.00 © 2007 Elsevier Inc. All rights reserved.
328
JOURNAL OF SURGICAL RESEARCH: VOL. 142, NO. 2, OCTOBER 2007
intestinal epithelial organoid units. Animals were housed in the Animal Research Facility at the Seattle Children’s Research Institute, Seattle, Washington, in accordance with National Institute of Health guidelines for the care of laboratory animals. Animals were maintained in a temperature-regulated environment (24°C) on a 12-h light-dark cycle, housed in cages with soft bedding and microisolator cover.
Polymer Scaffold Fabrication Microporous 3-D synthetic biodegradable polymer tubes were fabricated from nonwoven sheets of polyglycolic acid (PGA) fibers (fiber diameter 15 m; mesh thickness 2 mm; bulk density 60 mg/cm 3; porosity ⬎96%; mean pore size 250 m; Albany International, Albany, NY) as previously described [10]. The tubes were sprayed on the outer surface with a solution of polylactic acid (PLLA, Sigma) (5% wt/vol). The polymer tubes were sterilized with ethylene oxide, then coated with collagen Type 1 (Vitrogen; Cohesion Technologies, Palo Alto, CA). The scaffolds were rinsed with phosphate buffered saline (PBS) and Hanks balanced salt solution (HBSS) prior to cell seeding.
Perfusion Bioreactor Design and Fabrication The design for the perfusion bioreactor was adapted from the previously reported system [6]. The bioreactor consisted of a multichannel peristaltic pump, culture medium reservoir, oxygenation and gas exchange unit, air trap, and cell-polymer construct housing unit (Fig. 1). The culture medium was pumped at a flow rate of 1.5 mL/min from a 100 mL reservoir through the oxygenation and cellpolymer housing units and recirculated back to the reservoir. The adequacy of the oxygenation and gas exchange unit was assessed by measuring pH, PO 2, and PCO 2 of the culture medium with varying incubator CO 2 concentrations using a Bioprofile 400 Analyzer (NovaBiomed, Waltham, MA). The culture medium analysis was performed in duplicate (CO 2 10% and 15%) or triplicate (CO 2 5%) studies. The bioreactor circuit was modified using three-way stopcocks to allow the addition of a dynamic cell-seeding loop in continuity with the culture loop as well as sampling sites for culture medium analysis. The culture medium was collected distal to the cell-polymer housing unit for analysis. The entire circuit was sterilized by autoclave or ethylene oxide gas and maintained at 37°C with 8% CO 2 supplementation during cell seeding and culture. The bioreactor circuit was primed initially with PBS then with culture medium prior to loading with the cell-polymer constructs.
Cell Isolation Intestinal epithelial organoid units were harvested from nonfasted neonatal Lewis rats using an enzymatic dissociation technique as described by Evans [11]. Briefly, under isoflurane inhalational anesthesia, the entire length of small intestine was harvested, stripped of its mesentery, and placed in HBSS on ice. The intestines were flushed with cold HBSS, split open and cut into 2- to 3-mm fragments. The intestinal fragments were further washed with HBSS, sharply minced into ⬍1 mm 3 pieces, and then enzymatically dissociated (0.1 mg/mL dispase, neutral protease Type 1; Roche Applied Science, and 300 U/mL collagenase type 2, (Worthington) at room temperature on an orbital shaking platform at 80 rpm for 25 min. After mechanical agitation, the intestinal epithelial organoid units were further purified by centrifugation in a solution of Dulbecco’s modified Eagle’s medium, 2.5% heat-inactivated fetal calf serum, and 2% sorbitol (Sigma Chemical Co., St. Louis, MO) at 300 rpm for 2 min. The resulting pellet was resuspended in culture medium and counted.
Perfusion Bioreactor Culture The tubular polymer scaffolds were placed into the cell-polymer housing unit under sterile conditions after the circuit had been primed with PBS and culture medium. The intestinal epithelial organoid units were resuspended in culture medium and dynamically seeded through the seeding loop of the bioreactor circuit as previously described [6]. After the seeding period, the three-way stopcocks were turned to exclude the seeding loop, and the reservoir bottle containing the cell suspension was replaced with fresh warmed culture medium. The cell-polymer constructs (n ⫽ 4) were cultured under flow conditions for 2 d. Prior to harvest, the cells were incubated in 1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO) solution for an additional 12 h to assess general metabolic activity and distribution of cells within the scaffolds. The constructs were then harvested and processed for scanning electron microscopy and histology.
RESULTS Polymer Scaffold Fabrication
Highly porous tubular 3-D polymer scaffolds were fabricated with dimensions of 5 mm o.d., 2 mm i.d., and 10 mm length (Fig. 2). The devices were ⬎95% porous with a mean pore size approximately 250 m. The outer coating of PLLA provided structural integrity to the polymer tubes as previously reported [10]. Perfusion Bioreactor Design and Fabrication
A functional perfusion bioreactor culture system was successfully designed and manufactured (Fig. 3). Analysis of the culture medium under flow conditions demonstrated adequate oxygenation of the medium within the circuit. There was good correlation between culture medium pH and PCO 2 levels with changes in incubator CO 2 concentration demonstrating proper function of the gas exchange unit (Fig. 4). Intestinal Epithelial Organoid Unit Isolation FIG 1. Schematic diagram of the perfusion bioreactor. Dashed arrow depicts the seeding loop. Solid arrows depict the bioreactor loop.
Approximately 1.4 to 1.7 ⫻ 10 5 organoid units were harvested from each 7-d old neonatal Lewis rat. The organoid units were resuspended in culture medium at
KIM, PENKALA, AND ABRAHIMI: INTESTINAL TISSUE ENGINEERING
329
DISCUSSION
FIG 2. Highly porous biodegradable polymer tube made from polyglycolic acid fiber mesh. (A) Photomicrograph. (B) Low magnification scanning electron micrograph.
Short gut syndrome is a form of intestinal failure that has limited long-term treatment options. Tissue engineering approaches have been investigated as an alternative approach to treatment of intestinal failure and other forms of end stage organ disease. Our laboratory has been investigating the fabrication of functional living intestinal tissue using isolated cells combined with highly porous biodegradable polymer matrices. Previous studies in small animals have demonstrated the engraftment and survival of isolated intestinal epithelial organoid units transplanted on biodegradable polymer tubes, with regeneration of new tissue that morphologically resembled small intestine [1– 4]. Because of the difficulty in maintaining primary cultures of normal intestinal epithelium, researchers have developed methods of isolating intestinal epithelial organoid units, which preserve epithelial-mesenchymal interactions critical for epithelial proliferation and differentiation. Epithelial organoid units are also thought to contain intestinal stem cells and thus possess the capacity for regenerating new intestinal tissue. To rigorously investigate the mechanisms involved in intestinal epithelial proliferation, differentiation, and regeneration, we have hypothesized that a perfusion bioreactor system would be advantageous for in vitro culture and conditioning of intestinal cells as has been shown for other tissues [6 –9]. An in vitro system would allow for more uniform and reliable control of the environment in which the cells are initially exposed. It would minimize the period of hypoxia, which would potentially enhance cellular survival, proliferation, and differentiation. The effects of various trophic factors may be investigated in a more direct and precise manner than may be possible in an in vivo model. In addition, many investigators have demonstrated the beneficial effects of dynamic seeding tech-
a density of 2.5 ⫻ 10 4 organoid units/mL. Forty milliliters of the suspension was placed in the reservoir bottle and dynamically seeded through the seeding loop of the perfusion bioreactor. Perfusion Bioreactor Culture
After dynamic seeding and 2 d in flow culture, scanning electron micrograph demonstrated the successful attachment of intestinal epithelial organoid units onto the PGA polymer scaffold (Fig. 5). MTT assay demonstrated uniform distribution of viable cells throughout the polymer scaffold (Fig. 6). Histology confirmed the presence of viable clusters of cells with associated extracellular matrix within the PGA polymer scaffolds (Fig. 7). The cells from the organoid units appear to have coalesced into larger clusters and spread along the fibers of the scaffold, potentially as the initial steps in reorganization.
FIG 3.
Perfusion bioreactor culture system.
330
JOURNAL OF SURGICAL RESEARCH: VOL. 142, NO. 2, OCTOBER 2007
FIG 4. Culture medium analysis. Diamond: 5% CO 2 concentration; square: 10% CO 2 concentration; circle: 15% CO 2 concentration. (A) Culture medium PO 2 levels under varying incubator CO 2 concentrations. (B) Culture medium PCO 2 levels under varying incubator CO 2 concentrations. (C) Culture medium pH levels under varying incubator CO 2 concentrations.
niques in regards to cell attachment and uniform cell distribution within the polymer matrices [6, 12, 13]. In this study, we have been able to design and manufacture a functional perfusion bioreactor system for intestinal tissue engineering. This system can be sterilized and maintained under controlled conditions in vitro. It provides adequate oxygenation and CO 2 gas exchange. We have demonstrated that intestinal epi-
thelial organoid units can be dynamically seeded onto tubular polymer scaffolds and survive under flow conditions for 2 d. Further studies will be needed to optimize the parameters for dynamic seeding, optimize the polymer scaffold to enhance cellular attachment, investigate the long-term in vitro cultivation of intestinal cells seeded on biodegradable polymer scaffolds, and delineate the effects trophic factors for new tissue development. Continued efforts into long-term in vitro culture of intestinal epithelial and stem cells under
FIG 5. Scanning electron micrograph demonstrating successful attachment of intestinal epithelial cells onto the biodegradable polymer scaffold after dynamic seeding and 2 d of flow culture.
FIG 6. MTT assay demonstrating the uniform distribution of viable cells on the polymer scaffold after seeding and 2 d of flow culture.
KIM, PENKALA, AND ABRAHIMI: INTESTINAL TISSUE ENGINEERING
331
gradable polymer scaffolds for tissue engineering of small intestine. Transplantation 1999;67:227.
FIG 7. Hematoxylin and eosin histology demonstrating the presence of viable cells within the polymer scaffold 2 d after dynamic seeding and flow culture (magnification ⫻40).
flow conditions may yield important insights into the mechanisms involved in tissue morphogenesis and regeneration, and ultimately lead to the development of functional replacement tissues for the treatment of short gut syndrome. ACKNOWLEDGMENTS This work was funded by generous grants from the Department of Surgery, Children’s Hospital and Regional Medical Center and University of Washington, and the Seattle Children’s Hospital Steering Committee Award.
REFERENCES 1.
Kim SS, Kaihara S, Benvenuto MS, et al. Regenerative signals for intestinal epithelial organoid units transplanted on biode-
2.
Kaihara S, Kim SS, Benvenuto MS, et al. Successful anastomosis between tissue-engineered intestine and native small bowel. Transplantation 1999;67:241.
3.
Kim SS, Kaihara S, Benvenuto MS, et al. Effects of anastomosis of tissue-engineered neo-intestine to native small bowel. J Surg Res 1999;87:6.
4.
Kaihara S, Kim SS, Kim BS, et al. Long-term follow-up of tissue-engineered intestine after anastomosis to native small bowel. Transplantation 2000;69:1927.
5.
Kim SS, Utsunomiya H, Koski JA, et al. Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels. Ann Surg 1998;228:8.
6.
Kim SS, Sundback CA, Kaihara S, et al. Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system. Tissue Eng 2000;6:39.
7.
Portner R, Nagel-Heyer S, Goepfert C, et al. Bioreactor design for tissue engineering. J Biosci Bioeng 2005;100:235.
8.
Abousleiman RI, Sikavitsas VI. Bioreactors for tissues of the musculoskeletal system. Adv Exp Med Biol 2006;585:243.
9.
Holtorf HL, Jansen JA, Mikos AG. Modulation of cell differentiation in bone tissue engineering constructs cultured in a bioreactor. Adv Exp Med Biol 2006;585:225.
10.
Mooney DJ, Mazzoni CL, Breuer C, et al. Stabilized polyglycolic acid fibre-based tubes for tissue engineering. Biomaterials 1996;17:115.
11.
Evans GS, Flint N, Somers AS, et al. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J Cell Sci 1992;101:219.
12.
Vunjak-Novakovic G, Obradovic B, Martin I, et al. Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog 1998;14:193.
13.
Kim BS, Putnam AJ, Kulik TJ, et al. Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng 1998;57:46.