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Small intestinal submucosa: asubstrate for in vitro cell growthStephen F. Badylak a , Rae Record b , Kristina Lindberg c ,Jason Hodde d & Kinam Park ea Hillenbrand Biomedical Engineering Center, PurdueUniversity, A. A. Potter Bldg., West Lafayette, IN 47907,USAb Hillenbrand Biomedical Engineering Center, PurdueUniversity, A. A. Potter Bldg., West Lafayette, IN 47907,USAc Hillenbrand Biomedical Engineering Center, PurdueUniversity, A. A. Potter Bldg., West Lafayette, IN 47907,USAd Hillenbrand Biomedical Engineering Center, PurdueUniversity, A. A. Potter Bldg., West Lafayette, IN 47907,USAe Industrial and Physical Pharmacy, Purdue University,Robert Heine Pharmacy Bldg., West Lafayette, IN 47907,USAPublished online: 02 Apr 2012.

To cite this article: Stephen F. Badylak , Rae Record , Kristina Lindberg , Jason Hodde &Kinam Park (1998) Small intestinal submucosa: a substrate for in vitro cell growth, Journalof Biomaterials Science, Polymer Edition, 9:8, 863-878, DOI: 10.1163/156856298X00208

To link to this article: http://dx.doi.org/10.1163/156856298X00208

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Small intestinal submucosa: a substrate for in vitro

cell growth

STEPHEN F. BADYLAK 1,*, RAE RECORD1, KRISTINA LINDBERG1, JASON HODDE1 and KINAM PARK 2

1Hillenbrand Biomedical Engineering Center, Purdue University, A. A. Potter Bldg., West Lafayette, IN 47907, USA

2 Industrial and Physical Pharmacy, Purdue University, Robert Heine Pharmacy Bldg., West Lafayette, IN 47907, USA

Received 22 November 1997; accepted 9 February 1998

Abstraet-The extracellular matrix (ECM) of the small intestinal submucosa (SIS) was harvested by removing the superficial layers of the mucosa and the external muscular layers. The remaining 80 µm thick sheet was disinfected and sterilized by methods which removed all cellular components. The SIS-ECM, retaining its native 3-dimensional microarchitecture and composition, was evaluated for its

ability to support in vitro cell growth. Six separate cell types were seeded either alone or in coculture with other cells upon this matrix, grown in selected media, and examined daily for time periods ranging from 48 h to 2 weeks. The six cell types tested were NIH Swiss mouse 3T3 fibroblasts, NIH 3T3/J2 fibroblasts, primary human fibroblasts, primary human keratinocytes, human microvascular endothelial cells (HMECs), and an established rat osteosarcoma (ROS) cell line. All cell types showed the ability to attach and proliferate. All fibroblast cell lines and the keratinocytes proliferated and/or

migrated into the 3-dimensional scaffold of the SIS matrix. The ROS cells and the HMECs were confined in their growth pattern to the surface of the matrix. Coculturing of NIH 3T3/J2 fibroblasts and primary human keratinocytes resulted in a distinctive spatial orientation of the two cell types. The fibroblasts populated the mid-substance of the 3-dimensional matrix and the keratinocytes formed an

epidermal structure with rete ridge-like formation and stratification when the composite was lifted to an air liquid interface in culture. In summary, SIS provides a substratum with a 3-dimensional scaffold that allows for cell migration and spatial organization. This substratum is suitable for in vitro studies of the interaction between epithelial or mesenchymal cells and a naturally occurring extracellular matrix.

Key words: Small intestinal submucosa; cell culture; in vitro cell growth; cell growth substrate; substratum; extracellular matrix.

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INTRODUCTION

The attachment, growth, and differentiation of cells in vitro is critically dependent

upon the composition of the growth substrates within the culture media and within

the substratum upon which the cells are grown. Components of the in vivo

extracellular matrix (ECM) such as fibronectin, collagen, glycosaminoglycans,

hyaluronic acid, and fibrinogen have been purified and combined in various ways that are suitable for the growth of various cell types. Three-dimensional matrices

have also been developed which allow for the in vitro growth of cells not only on

the surface of the matrix but also within the matrix itself [l, 2]. A three-dimensional

substratum permits the migration and spatial orientation of cells in a manner which

theoretically more closely mimics the in vivo setting. Scaffolds which are derived

from naturally occurring ECMs and which have been used as cell culture substrates

include amnionic membrane [3], lens capsule [4], and basement membrane extracts

derived from Engelbreth Holm Swarm tumor (Matrigel). These matrices provide an

alternative substratum for cell growth and differentiation compared to the plastic cell

culture dish; however, their use has been limited by problems related to sterilization,

manufacturing, and availability. The field of tissue engineering has recently identified several biomaterials suitable

for use as tissue scaffolds. The materials are both synthetic and biologic in

nature, upon which cells are grown for the purpose of reconstituting or replacing various body parts. One such scaffold, derived from the submucosa and basilar

mucosal layers of the small intestine (SIS), has been shown to have properties which promote tissue remodeling in numerous body locations including the lower

urinary tract [5-7], dermis and epidermis [8], tendon and ligament [9, 10], body wall [11], and blood contact surfaces such as small and large diameter arteries

and veins [12-15]. This naturally occurring ECM consists of approximately 90%

collagen (mainly Type I), fibronectin [16], five different glycosaminoglycans [17],

growth factors such as TGFB and basic FGF [18], and various proteoglycans and

glycoproteins. The complex composition of SIS has been retained in its native

3-dimensional form for in vivo applications. Within 2-3 months of in vivo

grafting, the SIS scaffold is typically replaced by a host derived neomatrix with

cellular components which include fibroblasts, endothelial cells, and epithelial cells

appropriate for the recipient tissue. It is plausible therefore that the SIS biomaterial, with its natural 3-dimensional architecture would be useful for the evaluation of the

interaction between various cell types and the ECM in vitro. In the present study we evaluated the mode of growth of six separate cell types upon SIS in vitro.

MATERIALS AND METHODS

Preparation of'SIS

SIS was prepared from the jejunum of pigs weighing between 110 and 120 kg

according to methods previously reported [12-16]. In brief, intestines were

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collected immediately following euthanasia of the animals, rinsed free of contents

with water, and the superficial mucosal layers and external muscular layers were

removed by mechanical delamination. The remaining trilaminate tube structure

consisted of the submucosa, muscularis mucosa and basilar layers of the mucosa

(also called stratum compactum). This tube was split longitudinally creating a sheet

which was subsequently rinsed with water to lyse any remaining cells associated

with the matrix and rid the material of any cellular debris. A sheet of SIS was

oriented based upon identification of the stratum compactum surface. The opposite side of the SIS sheet was referred to as the abluminal surface. The tissue was then

disinfected with 0. I % peracetic acid in 20% ethanol followed by treatment with

1.5 MRad gamma irradiation. The sheets of SIS were then cut into appropriate size

and shaped to fit the tissue culture vessel.

Cell types grown on SIS

The following cell types were grown on sheets of SIS: Primary human fibroblasts

derived from a skin biopsy obtained from a clinically normal 42 year old woman

undergoing plastic surgery; NIH 3T3/J2 fibroblasts, a gift from Howard Green, Harvard Medical School, Boston, MA, USA; NIH Swiss 3T3 mouse fibroblasts, American type culture collection (ATTC) Rockville, MD; Human microvascular

endothelial cells (HMEC-1), a gift from Francisco Candal and colleagues at the

National Center for Infectious Diseases at the Center for Disease Control, Atlanta,

GA, USA; an established osteosarcoma cell line (ROS 17/2.8), a gift from Gregg Wesolowski at Merek, Sharp & Dohme Research Laboratories, West Point, PA,

USA; and primary cultures of human epidermal keratinocytes derived from a skin

biopsy obtained from a clinically normal 20 year old male.

Tissue culture conditions

The cell types and medium conditions used are described in Table 1.

Coculture of 3T3/J2 fibroblasts and primary human keratinocytes. Swiss mouse

NIH 3T3 /J2 were seeded at 5 x 106 cells cm-2 on the abluminal surface of NIH 3T3/J2 were seeded at 5 x 10? cells on the abluminal surface of

SIS. The fibroblasts on SIS were cultivated in Dulbecco's minimal essential

medium (DMEM) supplemented with 10% bovine calf serum (Hyclone, Logan, UT, USA) for 2-3 days prior to adding human epidermal keratinocytes to the

stratum compactum surface of SIS. The keratinocyte population was expanded in

the secondary or third passage using the Rheinwald and Green technique based on

cocultivation of Swiss mouse NIH 3T3/J2 cells and keratinocytes [19, 20]. The

tissue culture vessels were pre-plated with gamma-irradiated (6000 Rad) 3T3/J2-

fibroblast feeder cells (2.7 x 104 cells cm-2). The keratinocytes were seeded at

5 x 104 cells per 75 cm2 flask or at 0.5 x 106 cells per 75 cm2 flask (Corning Costar,

NY, USA) and grown in a 3 : 1 mixture of DMEM and Ham's F12 media (Sigma Chemical Co., St. Louis, MO, USA), supplemented with 10% fetal calf serum

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Table 1.

Summary of cell types and medium used

Cell type Medium'

Human DMEM and Ham's F12 media

epidermal 3 : 1 supplemented with 10% fetal

keratinocytes calf serum, 0.4 ng ml - 1

hydrocortisone, 10 f.1g ml-l human

epidermal growth, 10 IIM cholera toxin, 5 f.1g ml-l Zn-free

insulin, 24 f.1 g ml-1 adenine, 2 x 10-9 M 3,3',5-triiodo-L-thyronine

Human DMEM supplemented with 10% fibroblasts bovine calf serum

Swiss mouse DMEM supplemented with 10% NIH 3T3 neonatal calf serum fibroblasts

Swiss mouse DMEM supplemented with 10% NIH 3T3/12 bovine calf serum fibroblasts

Human MCDB 131 supplemented with microvascular 10% fetal bovine serum, 1 ng ml-l endothelial cells epidermal growth factor (EGF), (HMEC) and 1 /lg ml-1 hydrocortisone

Rat osteo- Ham's F-12 supplemented with sarcoma cells 28 mM HEPES and 1.1 mum

(ROS 17/2.8) Ca2+ and 5% fetal bovine serum

G 5000 U penicillin/5000 ttg streptomycin was added to all media.

(Hyclone, Logan, UT), hydrocortisone (Sigma Chemical Co., St. Louis,

MO, USA), 10 ng ml-l human epidermal growth factor (Austral Biologicals, San

Ramon, CA, USA), 10-1° M cholera toxin (Sigma Chemical Co., St. Louis,

MO, USA), 5 ,?g ml-1 Zn-free insulin (a gift from Lilly Research Laboratories,

Indianapolis, IN, USA), 24 ttgml-l adenine (Sigma Chemical Co., St. Louis,

MO, USA), and 2 x 10-9 M 3,3',5-triiodo-L-thyronine (Sigma Chemical Co., St.

Louis, MO, USA). After 6-7 days, preconfluent primary keratinocyte cultures were

incubated briefly with a 0.02% EDTA solution to remove the feeder cells. The

keratinocyte colonies were then dissociated into single cells by incubating the cells

with 0.05% trypsin and 0.01% EDTA PBS buffered solution and were subcultured

onto the stratum compactum surface of SIS at a density of 5 x 106 cells cm-2 SIS.

The NIH 3T3/J2 fibroblasts/keratinocyte/SIS composite was grown submerged in medium for 2-3 days, then lifted to the air-liquid interface. Alternatively, the keratinocytes seeded at the higher density which had reached confluence after

6-7 days were released as a cohesive sheet of cells by incubating the culture

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for 30-40 min in 2.5 mg ml-1 Dispase II (Boeringer-Mannheim, USA). The cell

sheet was mounted on a petroleum gauze support, 'transplanted' onto the stratum

compactum surface of SIS, and placed at the air-liquid interface. The composites

consisting of fibroblasts and keratinocytes on SIS were grown for 48 h, 5 days or

one week.

NIH Swiss mouse 3T3 fibroblasts and primary human fibroblasts. The NIH

Swiss mouse 3T3 fibroblasts and the human primary fibroblasts were maintained

in DMEM media supplemented with 10% neonatal calf serum and 10% bovine calf

serum, respectively. The fibroblasts were seeded on the abluminal surface or on the

luminal surface of SIS at 5 x 105 cells CM-2 SIS and grown for 48 h, 72 h, or for

2 weeks.

Human microvascular endothelial cells. HMEC cells were grown in MCDB 131 1

(Life Technologies) supplemented to contain 10% fetal bovine serum, 1 ng ml-1

epidermal growth factor (EGF), and 1 /-tgml-l hydrocortisone. The HMEC cells

were seeded on the abluminal surface or on the stratum compactum surface of SIS

at 5 x 105 cells cm-2 SIS and grown for 48 h, 72 h, or for 2 weeks.

Rat osteosarcoma cells. Rat osteosarcoma (ROS 17/2.8) cells ROS cells were

maintained in Ham's F-12 media modified to contain 28 mM HEPES and 1.1 mlvl

Ca 2+ and supplemented to contain 5% fetal bovine serum. The ROS cells were

seeded on the abluminal surface or on the stratum compactum surface of SIS at

5 x 105 cells cm-2 SIS and grown for 48 h, 72 h, or for 2 weeks.

Histological evaluation

The composites consisting of the various cell types on SIS were fixed in 10% neutral

buffered formalin at the time points stated above and embedded in paraffin. The

composites were then sectioned, stained with Hematoxylin and Eosin (H & E), and

evaluated using light microscopy.

RESULTS

Human epidermal keratinocytes cocultured with NIH 3T3/J2 fibroblasts

Human epidermal keratinocytes seeded as single cells (Figs 1 and 2) or 'trans-

planted' as a cohesive sheet of stratified keratinocytes onto the stratum compactum side of SIS (Fig. 3) showed attachment and changes in cell morphology. After 48 h

in culture the keratinocytes seeded as single cells had formed a monolayer (Fig. 1), and the basal cells of the cell sheet that had been 'transplanted' onto SIS were at-

tached to the substrate (not illustrated). By days 5 and 7 the upper strata of the

'transplant', which is up to six cell layers thick, had stratified and the disorganized

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cell sheet had terminally differentiated, forming a stratum comeum-like structure

(Fig. 3). At days 5 and 7 the epithelial-like structure, which was formed from the

keratinocytes seeded as single cells and the cell sheet that had been 'transplanted' onto SIS, consisted of a three to six cell layer thick stratified epithelium. The basal

cells tended to migrate into the SIS in a rete ridge-like pattern (Figs 2 and 3).

Human fibroblasts and NIH 3T3 fibroblasts

The three types of fibroblasts tended to form monolayers on the stratum compactum side of SIS. After 48 h in culture the fibroblasts seeded onto the abluminal surface

of SIS showed early migration into the substrate (Figs 4 and 5). Invasion into the

deeper layers of SIS usually did not occur before 72 h. By day 7, the fibroblasts

were often distributed throughout the 80 itm thickness of the SIS substrate (Fig. 5). The human fibroblasts showed an elongate, spindle shaped morphology consistent

with the usual morphological feature of fibroblasts. The NIH 3T3 fibroblasts were

larger and more plump than the human fibroblhsts. The cells remained elongate with

a centrally placed round oval nucleus. Cells could be found in both small clusters

as well as single cells which had migrated into adjacent areas of the substratum.

Human microvascular endothelial cells

The HMEC cells (Fig. 6) formed a monolayer at all time points studied. These cells

did not invade the SIS substratum when placed on either the stratum compactum surface or the abluminal surface. The HMECs maintained a spindle shaped

morphology with a centrally placed oval nucleus. There was no evidence for tube

formation.

Rat osteosarcoma cells

The ROS cells proliferated and showed changes in morphology when placed on

the SIS material (Fig. 7). The cells remained confined to the surface of the SIS

sheets regardless of whether they were seeded onto the stratum compactum or

the abluminal side of the SIS. The ROS cells grew in polylayers of three to six

cells thick. Small clumps of cells formed layers as thick as eight to ten cells.

The most superficial layers of the ROS cells showed a change in morphology. These superficial cells tended to become larger, occasionally multinucleated and

these cells contained vacuoles within their cytoplasm. In addition, there was

the appearance of an eosinophilic staining matrix around some of the cells in

both the superficial layers of the SIS and at the stratum compactum surface after

approximately 7 days.

DISCUSSION

This study demonstrates the utility of a naturally occurring extracellular matrix derived from the small intestine of pigs as a substratum for in vitro cell growth. Six

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separate cell types of mesenchymal and epithelial origin showed variable patterns of growth both on the surface and within the midsubstance of this substratum.

The advantages of SIS for in vitro cell growth include a composition and a

microarchitecture which is conducive to cell growth and allows for cell migration in

three dimensions. The composition and structure of SIS contrast markedly with

the majority of in vitro culture substrates upon which cells are grown in which

only two dimensional growth is possible. These two-dimensional systems usually involve an artificial substratum of plastic or glass. For example, cells have been

successfully grown on polyvinyl chloride, polycarbonate, polytetrafluoroethylene, melinex, and thermanox. Cells cultured upon these artificial substrates therefore

acquire their nutrition and signals for growth and differentiation almost entirely from the media in which they are grown and microenvironmental conditions such

as C02 concentration, barometric pressure, and temperature. In contrast, the SIS

substratum provides numerous structural characteristics which represent the native

environment encountered in vivo. Although it has been shown that SIS is rich in

growth factors, glycosaminoglycans and other potential nutrients for cell growth, the methods by which the present study were conducted with complete media makes

it impossible to determine the extent to which the composition of the SIS substratum

itself contributed to cell proliferation and differentiation. One limitation of the use

of the sheet form of SIS for in vitro cell culture is the inability to visualize the cells

due to the density of the substrate. Fixation of the substrate with attached cells and

subsequent sectioning and staining is necessary to obtain information regarding the

pattern of cell growth and/or degree of differentiation.

By providing a naturally occurring ECM such as SIS for in vitro cell culture, it is plausible that a pattern or mode of cell growth will more closely mimic the

in vivo behavior of such cells. For example, all three types of fibroblasts which

were evaluated grew throughout the entire thickness of the SIS matrix. The ROS

cells confined their growth to the surface but formed polylayers which maintained

distinctive phenotypic characteristics even in the most superficial cells. ROS cells, a

transformed osteoblast cell line, grow in a pattern reminiscent of osteoblasts in vivo

whereby new bone formation is laid down on the surface of existing bone and

connective tissue, similar to the cell growth pattern observed on SIS. Similarly, the pattern of keratinocyte growth into the underlying SIS substratum in the present

study is similar to the rete ridge formation which occurs in normal epidermis. The

major difference between the in vitro microenvironment with SIS and the in vivo

environment is the source of nutrition. In vivo, blood vessels would be supplying the substrates and nutrients for cell growth and differentiation whereby in vitro the

source of nutrition is provided by the media and/or substratum.

The biologic origin of SIS and its function in vivo likely contributes to its

utility as an in vitro substrate for cell growth. The complex composition of this

naturally occurring extracellular matrix is largely responsible for the in vivo signals which cause cell attachment, migration, proliferation, and differentiation. In vivo, the submucosa and basilar layers of the mucosa support a rapidly dividing cell

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population, the small intestine enterocytes. In addition, this layer is repeatedly

subjected to the insults originating in the intestinal lumen including infectious

agents, mechanical trauma, and digestive juices which occasionally breach the

mucosal barrier. Therefore, it is plausible that the extracellular matrix in this

location has developed a very suitable composition and structure for rapid cell

growth and differentiation (i.e. repair and regeneration). In summary, we report the utility of a naturally occurring extracellular matrix

derived from porcine small intestine for in vitro cell growth. This matrix provides a plethora of structural and functional proteins which are arranged in a three-

dimensional architecture which is suitable for examining cell growth patterns in vitro. The substrate is limited by its inability to permit visualization of cells

in vitro but offers a three-dimensional microarchitecture for cells to grow in a

more natural spatial arrangement than is possible with traditional two-dimensional

culture systems. SIS provides a complement to existing substrates for in vitro cell

growth, and may find utility in research laboratories where interest in cell-matrix

interactions exist.

Acknowledgements

The authors would like to acknowledge the technical assistance of Stephanie Hill,

Cheryl Haines, and Cheryl Holdman.

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