JP2002531118A - Applying shear flow stress to smooth muscle cells to produce implantable structures - Google Patents

Abstract

The present invention relates to a method of expanding smooth muscle cells in culture for the manufacture of tissue-treated implants or other implantable alternative structures. More specifically, the present invention relates to the application of shear flow stress to smooth muscle cells in culture. In this application, the cells are aligned perpendicular to the direction of flow, thereby bringing the cell orientation closer in vivo. The resulting cultures and methods are useful in the manufacture of improved vascular grafts, blood vessels and other implantable structures for correction of defects or abnormal tissue in the body.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is a pending provisional application filed on Dec. 11, 1998, US Provisional Application No.
Claims the benefits of 35 U.SC § 119 (e) of 60 / 111,760. The application is incorporated herein by reference in its entirety.

[0002] 1. INTRODUCTION The present invention relates to a method of expanding smooth muscle cells in culture for the manufacture of tissue-treated implants or other implantable alternative structures. More specifically, the invention relates to the application of shear flow stress to smooth muscle cells in culture. In this application, the cells are aligned perpendicular to the direction of flow, thereby allowing in vivo
Bring the cell orientation closer to o. The resulting cultures and methods are:
Improved vascular grafts for the repair of defects or abnormal tissues in the body,
Useful for the manufacture of tubes and other implantable structures.

[0003] 2. BACKGROUND OF THE INVENTION Vascular grafts or replacement vessels are used to repair or replace arterial and venous vessels weakened, damaged or occluded by trauma or disease (such as aneurysms, atherosclerosis and diabetes mellitus). Used by surgeons. Historically, vascular grafts have been identified as allografts such as the patient's own saphenous vein or internal thoracic artery, polyester (eg, Dacron), effervescent polytetrafluoroethylene (ePTFE).
) And other composite materials, such as prosthetic implants, or fresh, fixed or processed biological tissue implants have been used (eg, Vascular Surgery, 4th edition, Haimovici et al. (Eds.), Chapter 10, pp. 158-191
(See Blackwell Science, Inc. 1996).

[0004] In general, however, while synthetic grafts have inadequate patency for many applications, harvesting allografts is time consuming, expensive, and traumatic to the patient. Is necessary in excess. Fixed tissue grafts do not allow infiltration and colonization by host cells, which are essential for remodeling and tissue maintenance. As a result, the immobilized tissue graft will degrade over time and will eventually fail.

[0005] Due to the inadequacies of these currently available synthetic and biological grafts, and the cost and limited supply of allografts, tissue-treated grafts are not suitable for use in replacement therapy. Has been developed. See, for example, US Pat. No. 5,792,603, which is incorporated herein by reference in its entirety. The patent discloses an apparatus and method for culturing a vascular graft obtained by treating a tissue. Grafts treated with such tissue exhibit long-term dimensional stability and natural arterial and vascular patency, even more similar to normal physiological function. For example, Shinoka et al., Mar. 1998,
J. Thorac. Cardiovasc. Surg. 115 (3): 536-545. Also, U.S. patent application Ser.
See Issue 49. The application has recently been acknowledged and is incorporated herein by reference.

[0006] In forming cultures of these tissues, it is possible to grow appropriate cells or combinations of cells corresponding to biological structures requiring repair or replacement. For arterial repair or replacement, for example, it is necessary to use smooth muscle cells and / or endothelial cells. This is because in vivo arteries are muscle tubes lining up in a thin layer of endothelial cells. More specifically, a blood vessel is composed of three layers, namely, an inner layer, an intermediate layer, and an adventitia layer in order from inside to outside. For example, Textbook of Histology and Color Atlas of Histology
Second edition, 1986, Finn Geneser (eds.), Lea and Febiger (Philadelphi
a PA). The major cellular component of the inner layer is endothelial cells, and the major cellular component of the middle layer is smooth muscle cells. The smooth muscle cells produce most of the extracellular matrix of vascular tissue, such as proteins (such as elastin), proteoglycans, glycoproteins and collagen (eg, Thie et al., 1991, Eur. J. Cell Biol. 55:
295-304). In the artery, the inner elastic lamina is located inside the inner layer and outside the middle layer and is a homogeneous layer of fenestrated elastin. By the rich elastin in the arterial wall,
The ability of the arteries to expand and contract with each contraction of the heart is provided. The vein may also have heterogeneous or individual fibers of elastin in the vein wall. The adventitia is composed mainly of fibroblasts, more usually the vasculature (vasa vasorum) and some nerve tissue. The adventitia of the artery (but not the vein) also has a homogeneous and fenestrated outer elastic plate. Collagen and additional individual elastin in this layer are important for anchoring blood vessels. Veins are also tubular structures lined within endothelial cells, but the connective tissue layers are less well understood than the layers of arteries and their walls are thinner. Also, human veins lack internal and external elastic lamina. Thus, tissue treatment of vascular grafts can include culturing smooth muscle cells, endothelial cells and / or fibroblasts.

[0007] Historically, culturing of vascular grafts and tissues has generally been performed in a static environment, such as a petri dish or culture dish. However, in such a static environment, the circulation of nutrients required for proper growth can be impaired, resulting in slow and ineffective cell growth. In addition, cells cultured in a dynamic environment are exposed to conditions closer to the natural in vivo environment, which makes these cultured cells permissive for the physiological conditions that are implanted and exposed in the body. Likely to have

[0008] In fact, several studies have studied applying various mechanical pressures and hemodynamic forces to the growth of vascular wall cells in cell culture in vitro. For example, Benbrahim et al. (1996, J. Surg. Res. 65: 119-127) describe a vascular stimulator (VSD) in which human endothelial cells, bovine endothelial cells and bovine smooth muscle cells are tubulated. The structures were cultured on coated and uncoated silicone rubber to study the effects of arterial pressure, flow and strain conditions. The application of mechanical pressure or strain to the in vitro proliferation of vascular wall cells (such as endothelial cells and / or smooth muscle cells) has also been described by Mills et al., 1997, J. Cell Physiol. 170 (3): 228-234; Takemasa et al. , 1997
Exp. Cell Res. 230: 407-410; Kanda et al., 1994, Cell Transplantation 3 (6).
: 481-492; Kanada et al., 1993, ASAIO J. 39: M561-M565; Kanda et al., 1992, ASAIO.
J., 38 (3): M382-M385; and Buck et al., 1983, Artherosclerosis 46: 217-22.
Researched by three.

[0009] Shear flow stress is another type of pressure and its effect on vascular wall cells in vitro has been studied. For example, it is known that endothelial cells align parallel to the direction of shear flow stress in culture (eg, Ziegler et al., 199).
5, Cells and Materials 5 (2): 115-124; Levesque et al., 1985, J. Biomech. Eng.
107: 341-347; and Remuzzi et al., 1984, Biorheology 2: 617-630). Studies have also shown that endothelial cell alignment is dependent on multiple cellular pathways, such as intracellular calcium-dependent pathways and microtubule reorganization (eg, Malek et al., 1996, J.
Cell. Sci. 109: 713-726).

[0010] Shear flow stress has been shown to affect vascular smooth muscle cells in the following manner. That is, (1) proliferation of smooth muscle cells (Ueba et al., 1997, Arterioscl
er. Thromb. Vasc. Biol. 17: 1512-1516; Papadaki et al., 1996, Biotech. Bioeng.
50: 555-561; and Sterpetti et al., 1992, J. Cardiovasc. Surg. 33: 619-624.
) And reduces DNA synthesis (Sterpetti et al., 1992, supra); (2) causes the release of growth factors (such as PDGF, b-FGF and TGF-βl) by cells (Sterpe
tti et al., 1994, Eur. J. Vase. Surg. 8: 138-142 and Sterpetti et al., 1993, Surg.
113: 691-699); and (3) vasoactive substances (such as PGI2, PGE2 and NO)
(Papadaki et al., 1998, Am. J. Physiol. 274: H616-H626; and Alshihavi et al., 1996, Biochem. Biophys. Res. Comm. 224: 808-814).

However, the effect of shear flow stress on smooth muscle cell orientation or alignment is unclear. For example, Sterpetti et al. (1992, supra) show that by applying shear flow stress to bovine smooth muscle cells in vitro at 6 dynes / cm ² for 6 hours, the cells assumed a spherical morphology and aligned in the direction of flow. Is disclosed. In contrast, in vitro
There is also another clear statement that no selective alignment of smooth muscle cells was observed by exposure to shear flow stress in. For example, Malek et al. (1994, Circ. Res. 74
: 852-860) disclose that bovine aortic smooth muscle cells showed no altered cell shape or alignment in the direction of flow after 24 hours of exposure to shear flow stress (15 dynes / cm ² ). are doing. Similarly, Papadaki et al. (1996, supra) report 5 or 25
No alignment in the direction of flow of human smooth muscle cells after 24 hours in either dynes / cm ² was found.

[0012] Other studies have involved the co-culture of smooth muscle cells with endothelial cells and the application of shear flow stress to the endothelial cells. See, for example, Nachman et al., 1998, Surg. 124 (2): 353-361; and Ziegler et al., 1995, supra. Yes, studies analyzing the effects of shear flow stress directly on smooth muscle cells. In a final conclusion of the Ziegler et al., Supra, critics argue that when cultured on glass or plastic, smooth muscle cells are oriented perpendicular to the direction of flow. Has not performed such experiments and no publications or data in the art substantiate this comment. In addition, Ziegler et al. (Supra) and other Weinberg et al. (1985, J. Cell
Physiol. 122: 410-414) and Thie et al. (1991, Eur. J. Cell Biol. 55: 295-).
304) et al. Describe the growth of smooth muscle cells in three-dimensional collagen gels, but none of them disclose the analysis of the effect of shear flow stress on smooth muscle cell orientation or alignment.

[0013] However, it is known that smooth muscle cells align peripherally, ie, perpendicular to the direction of blood flow in blood vessels, in vivo (eg, natural blood vessels) (eg, Dartsch et al., 1986, Acta Anat. 125: 108-113 and Kanda et al., 1994.
, See above). This natural orientation may be necessary to provide sufficient mechanical pressure to the blood vessel for proper biological function under physiological pressure (eg, Tranquillo et al., 1996, Biomaterials 17: 349- 357; L'Heureux et al., 1993, J. Va
sc. Surg. 17: 499-509).

Thus, the method of culturing smooth muscle cells and the resulting tissue-treated cultures may result in the cells aligning in vivo in a manner more similar to the orientation of natural smooth muscle cells. And would be most useful for the production of more effective vascular grafts and other implantable alternative structures.

[0015] 3. SUMMARY OF THE INVENTION The present invention provides an in vitro method for the production of both monolayer and three-dimensional tissue processing cultures.
The application of shear flow stress to smooth muscle cell proliferation in ro. In this application, the cells are oriented or aligned perpendicular to the direction of flow. According to a preferred embodiment, the smooth muscle cells are subjected to shear flow stress in culture, with or without other cells (eg, fibroblasts and / or endothelial cells), for the manufacture of vascular grafts or replacement blood vessels. Proliferate at In this case, the smooth muscle cells align with the periphery in the implant, ie, perpendicular to the direction of flow. In other embodiments, the smooth muscle cells and / or fibroblasts are skeletal muscle, ligaments, tendons or chordae for reconstruction surgery,
The lining, wrap (ie, re-enforcement) or adaptation (
oriented) For the production of tissue sheets, they can be grown under shear flow stress in culture.

According to the method of the present invention, the smooth muscle cells are cultured in vitro with or without fibroblasts or other stromal cells and the components described below in two-dimensional (eg, monolayer) cultures or Inoculate and grow on a substrate in a three-dimensional culture. The cells can adhere to the substrate and are then subjected to shear flow stress for a time sufficient to induce vertical alignment of the smooth muscle cells with respect to the direction of the shear flow. Alternatively, cells may be cultured in vitro for a desired period of time, and then subjected to shear flow stress for a period of time sufficient to induce vertical alignment of smooth muscle cells to the direction of flow.

[0017] The shear flow is preferably stable and laminar, with a shear flow stress level of about 0.1 to about 10
0 dynes / cm ^2, more preferably from about 0.3 to about 50 dynes / cm ^2, and more preferably about
It can range from 0.3 to about 25 dynes / cm ² . After the application of shear flow stress, the smooth muscle cells
Depending on the applied pressure level, alignment may begin within hours or at most weeks. One of skill in the art will be able to experimentally determine the appropriate level of shear flow stress to apply after a desired time to obtain alignment. For example, according to one embodiment, the shear flow stress is applied at a shear flow stress level of about 20 dynes / cm ² for at least 24 hours, more preferably at least 48 hours. According to another embodiment, the shear flow stress is applied at a level of about 0.3 dynes / cm ² for about 3 weeks.

According to a further embodiment of the present invention, the smooth muscle cells are genetically modified by an exogenous gene. Preferably, the exogenous gene is under the control of a regulated promoter and expresses a gene product that is advantageous for successful and / or improved transplantation / implantation, functioning of the disease state, or improved maintenance. For example, in the case of vascular grafts, anti-inflammatory drugs that direct smooth muscle cells to express anticoagulant gene products that reduce the risk of thromboembolism, atherosclerosis or occlusion, or reduce the risk of tissue rejection It can be genetically engineered to express a gene product. The cells may be genetically engineered to produce "knock-out" expression of factors or surface antigens that promote coagulation or rejection. In addition, genetically modified cells produce factors through the control of shear flow stress (and possibly shear stress control elements), and the factors (delivered either locally or systemically) are responsible for other physiological functions. (Eg, preventing thrombosis or even producing insulin at a certain level).

According to another preferred embodiment, the smooth muscle cells are subjected to shear flow stress on a three-dimensional framework of biocompatible non-biological material, with or without other stromal cells (such as fibroblasts). To form a three-dimensional living tissue construct. When the construct is subjected to shear flow stress, the smooth muscle cells align perpendicular to the direction of flow, resulting in a three-dimensional smooth muscle cell tissue construct. In this case, the cells align in a manner more similar to natural blood vessels or structures.

In a further embodiment of the invention, the vertically aligned three-dimensional smooth muscle cell tissue construct is further inoculated with endothelial cells and shear flow stress is applied to the endothelial cells to induce a parallel alignment of the endothelial cells. To produce vascular grafts that are very similar to natural blood vessels. That is, in this case, the smooth muscle components align perpendicular to the direction of the shear flow, and the endothelial cell components align parallel to the direction of the flow.

[0021] Yet another embodiment of the method of the present invention is a method of culturing smooth muscle cells under shear flow stress in a monolayer culture and forming the aligned cells into a tubular structure to form an improved tubular vessel. Producing an implant. According to another embodiment of the method, a monolayer of vertically aligned smooth muscle cells is further inoculated with endothelial cells (either before or after tube formation) and induces a parallel alignment of endothelial cells. To apply shear flow stress to
An improved vascular graft containing at least one layer of smooth muscle cells and an inner layer of endothelial cells is produced.

The method of the present invention for culturing the smooth muscle cells of the present invention and the tissue constructs obtained therefrom is provided by an improved vascular graft and / or other repair or alternative structure for implantation or transplantation in vivo. It is useful for manufacturing products. Accordingly, embodiments of the present invention include improved in vitro prepared tubular structures for in vivo implantation, for example, a tubular tertiary of biocompatible non-biological material having voids cross-linked by stromal cells. Include a vascular graft containing stromal cells, including smooth muscle cells on the primary scaffold, wherein the smooth muscle cells align with the tubular structure on the surrounding scaffold. The tubular structure is further inoculated with endothelial cells and subjected to shear flow stress so that the endothelial cells line the lumen of the tubular structure and can be longitudinally aligned with the tubular structure. Such vascular grafts can include arterial or venous grafts. These improved tubular structures may be genetically modified by at least one exogenous gene described below.

3.1 Definitions The following terms used herein have the following meanings.

Adhesive layer: Cells that adhere directly to a three-dimensional support or are indirectly attached by attaching to cells that directly adhere to the support.

Stromal cells: Smooth muscle cells and / or fibroblasts, which may or may not have other cells and / or components found in loose connective tissue, which are defined Although not intended, endothelial cells, pericytes, macrophages, monocytes, plasma cells,
Mast cells, adipocytes, tan cells, myofibroblasts, stromal cells of Wharton's jelly and the like are included.

Tissue-specific cells or parenchymal cells: Cells that form an essential and distinctive organ tissue, distinguished from the supporting framework.

Three-dimensional scaffold or scaffold: a three-dimensional support structure composed of any material and / or shape, such that (a) cells can be attached to it or cells can be attached to it And (b) permit the cells to grow in one or more layers. The support is inoculated with stromal cells to form a living three-dimensional stromal matrix. For example, the framework structure may include, but is not limited to, a mesh or sponge, and may be formed in the form of a tube.

Biological stromal matrix: A three-dimensional framework inoculated with stromal cells grown on a support.
The extracellular matrix proteins synthesized by the stromal cells are oriented on the scaffold, thereby forming a biological stromal tissue or matrix. The biological stromal matrix supports the growth of tissue-specific cells after inoculation and can form a three-dimensional cell culture.

Tissue-specific three-dimensional construct: A three-dimensional biostromal matrix inoculated and cultured with tissue-specific or parenchymal cells. Generally, the tissue-specific cells used to inoculate the three-dimensional stromal matrix need to contain "stem" cells (i.e., "reserve" cells) for the tissue, i.e., cells that produce new cells. There is. The cells will mature and become specialized cells that form the parenchymal or differentiated matrix of the tissue or a tissue-specific matrix.

5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing smooth muscle cells by applying shear flow stress to the cells. The present invention is also directed to a tissue construct prepared by treating tissue prepared from a monolayer or three-dimensional smooth muscle culture, to produce an improved vascular graft or other repair or replacement structure for implantation / transplantation. A tissue construct characterized in that the cells are properly aligned.

The present invention is based, in part, on the discovery that applying shear flow stress to a three-dimensional or monolayer culture of smooth muscle cells induces cell alignment perpendicular to the direction of shear flow. . It is well known that smooth muscle cells in blood vessels in vivo are found to be oriented in the circumferential direction, i.e., perpendicular to the blood flow through the blood vessels, so using the methods and constructs of the present invention, It is possible to create improved vascular grafts and other implantable structures that are more similar to the corresponding tissue in vivo.

As used herein, the term “shear flow stress” means the force generated by a fluid that acts on a cultured cell by the relative movement of the cell and a liquid, such as a liquid medium. The term "shear flow stress" is also referred to in the art as "fluid shear stress". Shear flow stress can be generated by moving a liquid past a stationary cell, moving a cell through a stationary liquid, or moving a liquid and a cell simultaneously. Small particles, such as beads or microspheres, may optionally be included in the liquid to increase the viscosity of the liquid and increase the level of shear (see Equation (1) in Section 5.2 below). I want to do that).

The shear flow stress is generally quantified by dynes / cm ² and is preferably steady and laminar. Level of shear flow stress is to take a value in the range from about 0.1 to about 100 dynes / cm ^2, more preferably from about 0.3 to about 50 dynes / cm ^2, more preferably from about 0.3 to about 25 dynes / cm ² Can be. After applying the shear flow stress, the smooth muscle cells can begin to align within hours or weeks, depending on the level of stress applied. One of ordinary skill in the art will be able to experimentally determine the appropriate level of shear flow stress to apply to achieve alignment after the desired time has elapsed. According to one embodiment, at a shear flow stress level of about 20 dynes / cm ² , the shear flow stress is applied for a duration of at least 24 hours, more preferably for a duration of at least 48 hours. According to another embodiment, a shear flow stress at a level of about 0.3 dynes / cm ² is applied for about 3 weeks.

According to the methods and constructs of the present invention, smooth muscle cells (differentiated and / or undifferentiated)
Are grown on substrates in vitro in two-dimensional or three-dimensional cultures, with or without endothelial cells, fibroblasts, and other connective tissue or stromal cells, and subjected to shear flow stress . As used herein, the term "substrate" refers to any surface to which cells can attach and grow. Two-dimensional cell cultures can contain a culture of cells as a monolayer or on a monolayer substrate. Subjecting such smooth muscle cell cultures to shear flow stress according to the present invention induces the smooth muscle cells to align in a direction perpendicular to the direction of flow.

According to a preferred embodiment, smooth muscle cells are seeded on a biocompatible non-living three-dimensional skeleton or scaffold support structure and subjected to shear flow stress. According to another preferred embodiment, smooth muscle cells and other stromal cells are cultured on a three-dimensional biocompatible non-living three-dimensional skeleton and subjected to shear flow stress. According to one such embodiment, the stromal cells, including smooth muscle cells and fibroblasts, are the third stromal cells described above.
As defined in section 1, the three-dimensional scaffold is inoculated with or without additional stromal cells or components. Preferably, the cells are mammalian cells, more preferably human cells, which are seeded on or in a three-dimensional scaffold and grow there to form a biostromal matrix.

The stromal cells used in this embodiment may be of embryo, fetal, adult, and appropriate organs, such as blood vessels, eg, arteries, veins, or umbilical cord or placenta. It may be derived from a tissue. Such tissues and / or organs can be obtained by appropriate biopsy, surgical disposal, organ harvesting, or at necropsy. In practice, cadaver organs may be used to supply large quantities of stromal cells and components. However, the use of cells from allogeneic sources can create problems associated with graft rejection due to immunological factors. Thus, in some cases, it may be preferable to use undifferentiated or embryonic or fetal stromal cells, such as fetal fibroblasts.
Such fetal cell sources include placenta and umbilical cord. The stromal matrix from the fetus may have “general” tissue properties that can be further governed by the addition of growth factors or by certain growth conditions.

In other cases, it may be preferable to use a “specific” stromal support matrix over a “general” stromal support matrix. In this case, a particular organization,
Stromal cells and components can be obtained from an organ or an individual. For example, if a three-dimensional culture is to be used for transplantation or implantation in vivo, stromal cells and components (eg, smooth muscle cells and Fibroblasts) may be preferred. This approach would be particularly advantageous where immunological rejection of the transplant and / or graft with the host disease is likely. Furthermore, smooth muscle cells,
Fibroblasts, as well as other stromal cells and / or components, can be from the same type of tissue to be cultured in a three-dimensional system. This would be advantageous when culturing tissues where differentiated stromal cells may play a specific structural / functional role, for example, arterial or venous smooth muscle cells.

When seeded on a three-dimensional scaffold, stromal cells grow on the scaffold and produce growth factors, regulatory factors, and extracellular matrix proteins that are deposited on the scaffold. Connective tissue proteins naturally secreted by stromal cells substantially surround the scaffold to form a biological stromal matrix or tissue construct. This biostromal matrix can further sustain the growth of tissue-specific cells inoculated on the matrix.

As noted above, smooth muscle cells may be inoculated on a non-biological skeleton with or without other stromal cells to form a biological stromal matrix. Alternatively, fibroblasts can be inoculated on the skeleton with or without smooth muscle cells to form a biostromal matrix, and then inoculated with smooth muscle cells on the biostromal matrix (As tissue-specific cells). In each case, these three-dimensional tissue constructs will sustain active growth of the culture over an extended period of time. Opening in the skeleton allows for the delivery of stromal cells in culture, so confluent stromal cultures do not exhibit contact inhibition and stromal cells continue to grow and divide and remain functionally active State is maintained. Thus, the three-dimensional systems described herein support maturation, differentiation, and segregation of cells in culture, i.e., in vitro, and form components of adult tissue similar to those found in vivo. .

Although growth and regulatory factors may be added to the culture, they are not required because they are produced by stromal tissue. To further promote colonization of the three-dimensional skeleton or scaffold, growth factors (such as, but not limited to, αFGF, βFGF, insulin growth factor, or TGF-β), or natural or modified blood products, or Other bioactive biomolecules (eg, but not limited to, hyaluronic acid or hormones) may be used, but are not absolutely necessary for the present invention.

According to the present invention, it is important that the cellular microenvironment found in vivo for a particular tissue be reformed in culture, so prior to using the culture in vivo,
The extent to which a biological stromal matrix or tissue-specific culture is grown depends on
It may vary depending on the type of tissue grown in three-dimensional tissue culture. Furthermore, by implanting in vivo, the biological matrix itself can be modified (
corrective structure). In addition, tissue-specific cells, such as smooth muscle cells and / or endothelial cells, are inoculated into a biological stromal matrix,
It may be implanted in vivo with or without in vitro pre-culture.

Furthermore, cells grown in the above-described system, such as matrix stromal cells or tissue-specific cells, can be transformed into gene products useful for transplantation, such as anti-inflammatory factors, specifically anti-inflammatory factors.
It may be genetically engineered to produce GM-CSF, anti-TNF, anti-IL-1, anti-IL-2 and the like. Alternatively, `` knock out '' the expression of natural gene products that promote inflammation, such as GM-CSF, TNF, IL-1, IL-2, or express MHC antigens to reduce the risk of rejection The cells may be genetically engineered to "knock out" the cells. In addition, cells may be genetically engineered for use in gene therapy, resulting in tissue transplantation or other physiological functions associated with vascular function,
For example, expression of nitric oxide to reduce patency and reduce thrombogenicity or expression of PAI-1 to reduce stenosis, or other physiological functions not associated with vascular function, e.g., The patient's gene activity level may be adjusted to assist or improve the expression of insulin.

In another alternative, the cells are genetically engineered to allow smooth muscle cells to proliferate, migrate, and, for example, by antisense oligodeoxynucleotide blockade of the expression of cell division cycle 2 kinase and proliferating cell nuclear antigen, and It can block gene expression required for the transition leading to neointimal hyperplasia. For details of the genetically engineered cells used in the methods and constructs of the invention, see:
See section 5.4.3.

Thus, according to the present invention, a three-dimensional construct as described above, including smooth muscle cells and / or fibroblasts and other stromal cells, is placed in a bioreactor, for example, to produce an improved vascular graft. At shear flow stress. For example, as shown in FIG. 7, a bioreactor includes a growth chamber in which a three-dimensional tissue construct is placed, a pump, a media reservoir, and a connecting tube. During culture, applying shear flow stress to the construct results in a vascular graft construct having smooth muscle cells oriented perpendicular to the direction of flow. This construct is more similar to the condition of smooth muscle cells in blood vessels in vivo, and thus will have greater potential to withstand the physiological conditions exposed in the human body upon implantation. In this way, the bioreactor creates a dynamic environment for culturing tissue-treated vascular grafts or other biografts or other implantable constructs.

For ease of explanation only, the detailed description of the invention is described in the following sections: (1) Monolayer and three-dimensional tissue constructs of the present invention (2) applying shear flow stress to the construct, (3) aligning smooth muscle cells under shear flow stress, and (4) using the methods and constructs of the present invention. .

5.1 Preparation of Monolayer and Three-Dimensional Constructs of the Invention 5.1.1. Cell Isolation According to the invention, endothelial cells, fibroblasts, and other connective tissue cells, for example,
Inoculation of monolayer or three-dimensional cultures with smooth muscle cells, with or without tendon cells, pericytes, plasma cells, mast cells, adipocytes, etc. The specific cells utilized will depend on the type of graft desired. For example, in the case of an arterial or venous graft, the inoculum may contain medial smooth muscle cells and may further contain adventitia or dermal fibroblasts and / or intima endothelial cells. . The source of these inoculum cells may vary depending on the implantable structure to be repaired or replaced. For example, if the implant or structure is for a small tube, these inoculum cells (eg, smooth muscle cells, fibroblasts, and / or endothelial cells) may be, for example, coronary, breast, tibia, fibula, It may be obtained from the artery and / or vein of the femoropopliteal, dorsal, tarsal, arcuate or plantar, umbilical cord blood vessels or placenta. If the graft is for a medium sized vessel, the cells may be obtained from a vessel such as, for example, a carotid, clavicular, or femoral artery. Where large blood vessels are involved, cells may be obtained, for example, from the aorta, pulmonary artery, thoracic artery, aortic iliac artery, or aortofemoral artery (or vein, where appropriate). If the repair or replacement structure is an internal organ, the stomach, intestines, uterus,
Alternatively, involuntary smooth muscle cells derived from an internal body organ such as the bladder can be used. Such involuntary smooth muscle cells can also be utilized as a cell source for grafts of vessels, such as blood vessels.

The smooth muscle cells (also referred to herein as “SMCs”), fibroblasts, and other connective tissue cells used in the constructs and methods of the present invention may be of the type well known in the art. Can be isolated using established methods. As mentioned earlier, these cells can be obtained from embryos, fetuses, adults, and biopsies (where appropriate).
Alternatively, it may be derived from various organs such as blood vessels, ligaments, or tendons that can be obtained at the time of necropsy.

More specifically, SMCs and fibroblasts can be easily isolated using techniques well known to those of skill in the art by dissociating a suitable organ or tissue that serves as a source of cells. It is possible. For example, tissues or organs can be subjected to mechanical dissociation treatment and / or treatment with digestive enzymes and / or chelators that weaken the bonds between adjacent cells, thereby causing no discernible cell destruction It is possible to disperse the tissue into a suspension of individual cells. Enzymatic dissociation can be achieved by mincing the tissue and treating the minced tissue with any of several digestive enzymes, alone or in combination. These enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, and / or hyaluronidase, DNase, pronase, dispase, and the like. Mechanical crushing is also achieved by several methods, including but not limited to the use of a grinder, blender, sieve, homogenizer, pressurized cell, or insonator, to name a few. be able to. For an overview of tissue dissociation methods, see Freshney, Culture of A.
nimal Cells. A Manual of Basic Technique, 2nd edition, AR Liss, Inc., New
See York, 1987.

Once the tissue has been subdivided into a suspension of individual cells, the suspension can be used to obtain SMCs or fibroblasts and / or other small cells from which stromal cells and / or components can be obtained. They can be separated into groups. This also includes, but is not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based on differences in cell aggregation in mixed populations, freeze-thaw processing Method,
Differences in cell adhesion properties in mixed populations, filtration, conventional and zonal centrifugation,
Achieved using standard methods of cell separation, including centrifugal elution (countercurrent centrifugation), unit gravity separation, countercurrent distribution, electrophoresis, fluorescence activated cell sorting (FACS), or LDL uptake assay Is possible. For an overview of clone selection and cell separation methods, see Freshney, supra, Chapters 11 and 12, pp. 137-168. See also Shinoka et al., Supra. For example, an antibody that binds to a cell surface marker of SMC or fibroblasts is coated on a tissue culture plate,
The antibody is used to selectively bind to each of those cells from a heterogeneous cell population. Similarly, SMC-specific or fibroblast-specific antibodies can be used in a FACS method to isolate SMC or fibroblasts, respectively.

The vascular SMC used in the methods and constructs of the present invention can be explanted (eg, fr
eshney, supra) or by collagenase digestion (Jaffe, 1973, J.
Clin. Inv. 32: 2745-2756), can be isolated from various arteries. More specifically, vascular SMCs are isolated by removing the intima and adventitia to expose the media. The inner tissue is minced, explanted, and incubated on tissue culture plastic in a minimum volume of nutrient medium with 20% FCS at 95% humidity, 5% CO ₂ , and 37 ° C. Similarly, the adventitia is treated to release arterial fibroblasts. Cell proliferation can generally be observed within 7-14 days. For a detailed example of the isolation of vascular SMC, see Section 6.1 in the Examples below.

In addition, for long unbranched arteries, a cannula may be inserted at either end,
Perfused saline to remove excess blood, and filled with SM cocktail containing collagenase, esterase, DNase, and soybean trypsin inhibitor
C can be released into the suspension. Similarly, the enucleated outer membrane can be enzymatically treated to release arterial fibroblasts.

[0052] Alternatively, fibroblasts may be isolated as follows. Fresh tissue samples are washed extensively and minced in Hank's balanced salt solution (HBSS) to remove serum. The minced tissue is incubated for 1-12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After such incubation,
Dissociated cells are suspended, pelleted by centrifugation, and plated on culture dishes. Fibroblasts typically attach before other cells, so that appropriate stromal cells can be selectively isolated and expanded. The isolated fibroblasts can then be grown to confluence, picked up from the confluent culture, and inoculated on a monolayer substrate or on a three-dimensional skeletal support (Naughton et al., 1987, J
Med. 18 (3 and 4): 219-250).

The inoculating endothelial cells in the cultures of the present invention can be isolated from tubes with an ischemic time of less than 3 hours using a tube size specific method. Large tubes are cut open to a length of 5 cm and flattened to expose the intimal surface (Gospodarowicz et al., 1976, Proc.
Natl. Acad. Sci. USA 73 (11): 4120-4124). The intimal surface is washed with calcium-free saline and a sterile swab is rolled over the endothelial surface with slight pressure. Spinning the swab releases only the endothelial cells into the nutrient medium containing 10% serum and bFGF. Next, the medium containing the cells is plated on a culture dish.

For shorter unbranched tubes, either end is cannulated, perfused with saline, and then filled with 0.02% collagenase solution and incubated for 10 minutes. The digest is removed and diluted with media containing 10% FCS and centrifuged to pellet endothelial cells. Plate cells in culture flasks in Medium-199 containing 10% FCS, antimicrobial agent, endothelial cell growth supplement, and heparin (e.g., Sorger, 1995, In Vitro Cell Dev. Biol.-Animal 31: 671 -683).

After isolation, the cells are seeded on a substrate to produce a tissue culture, and then subjected to shear flow stress according to the method of the invention.

5.1.2 Monolayer Cultures SMCs, fibroblasts, and / or endothelial cells isolated as described above can be used on various well-known substrates to make monolayer cultures of the invention. Can be inoculated. The substrate utilized can be made from any biocompatible material that allows cells to attach and grow in a monolayer. For example, non-porous scaffold structures made from biodegradable or non-biodegradable materials and the like may be used. Also, synthetic articles, such as glass, plastic, or metal substrates, or non-synthetic or biological artificial scaffolds (composed of collagen, elastin, or polymers), or composites, hybrids, or combinations thereof Cells can also be plated on top. The substrate may further be in the form of a culture dish, slide, flask, plate, drum, or disk.

According to one embodiment, the cells are seeded in roller bottles and then continuously expanded, trypsinized, counted, and then seeded on a biodegradable or bioresorbable scaffold. You may leave it overnight. The scaffold may have previously been disinfected with a 20X antibiotic / antimycotic solution or sterilized by irradiation and pretreated overnight with 10% BCS DMEM complete medium or 10 mg / ml fibronectin. Good. By stirring gently by direct inoculation or cell suspension of cells, for example, about 1 to 5 × 10 ⁶ cells / cm ^2, more preferably inoculated into the scaffold at about 3 × 10 ⁶ cells / cm ² ,
Subsequently, stationary culture can be performed in the bioreactor.

In section 6.2 of the Examples below, cultured SMCs (passage 6-8) are plated and confluent on glass slides (2 μg / cm ² fibronectin) coated with fibronectin. The monolayer culture was grown until. These monolayer cultures were then subjected to shear flow stress using a parallel plate flow chamber. Alternatively, the cells may be grown as a monolayer culture on the surface of a drum, plate, or disk and then placed in a bioreactor to apply shear flow stress to the monolayer culture. Is also good.

5.1.3. The three-dimensional constructs of the present invention or, as described above, SMCs, fibroblasts, and / or endothelial cells, with or without additional stromal components, By inoculating onto or into a three-dimensional scaffold or scaffold, a three-dimensional tissue construct to which shear flow stress is to be applied can be formed. For the parameters of the three-dimensional biostromal matrices and tissue-specific constructs of the present invention, their retention, and various uses of such cultures, U.S. Pat.No. 4,963,489, commonly owned, is hereby incorporated by reference. It is described in. Applicants have no obligation or responsibility to explain the mechanism of action of the present invention, but as noted below, several factors specific to three-dimensional culture systems may contribute to the success of the present invention. There is.

(A) The three-dimensional scaffold provides a larger surface area for proteins to bind and thus for stromal cells to adhere. Moreover, (b) because the skeleton is three-dimensional, the stromal cells continue to proliferate actively, in contrast to cells in monolayer culture. Monolayer cultures grow to confluence, exhibit contact inhibition, and stop growth and division. The production of growth and regulatory factors by replicating stromal cells may be partially involved in promoting cell growth and regulating cell differentiation in culture. (c) Using a three-dimensional scaffold, the spatial distribution of the cellular components is more similar to that found in the corresponding tissue in vivo. (d) As the volume in which cells can grow in a three-dimensional system increases, a localized microenvironment that contributes to cell maturation may be formed. (e) The three-dimensional skeleton maximizes cell-cell interactions by enhancing the motility of migrating cells such as fibroblasts and smooth muscle cells. (f) It has been confirmed that not only growth / differentiation factors but also appropriate cell interactions are required to maintain the phenotype of differentiated cells. The present invention efficiently reproduces the microenvironment of a tissue.

The three-dimensional support or scaffold can be (a) capable of binding cells (or modified to allow cells to bind), and (b) cells in two or more layers. Can be any material and / or shape that allows the growth of the skeleton. A wide variety of materials can be used to make the scaffold, including non-biodegradable materials such as nylon ( (Polyamide), Dacron (polyester), polystyrene, polypropylene, polyacrylate, polyvinyl compound (for example, polyvinyl chloride), polycarbonate (PVC), polyurethane, polytetrafluoroethylene (PTFE; Teflon (registered trademark)), Thermonox (TPX ), Nitrocellulose, cotton; and biodegradable substances such as polyglycolic acid (PGA), collagen, collagen sponge, intestine Sutures, including, but not limited to, cellulose, gelatin, dextran, polyalkanoate, polycaprolactone, and copolymers, all of which can be woven, braided, knitted, It is possible to form a three-dimensional skeleton by casting it together with the porogen and then leaching it into a scaffold structure, followed by deformation of the skeleton and a correction structure such as a tube, Can be in any desired shape, such as ropes, filaments, sponges, etc. Certain materials, such as nylon, polystyrene, etc., are substrates with poor cell binding. In some cases, it may be desirable to pre-treat the scaffold prior to inoculating the stromal cells to promote the binding of the stromal cells to the support. Before inoculating stromal cells, the nylon scaffold can be treated with 0.1 M acetic acid and coated with nylon by incubating in polylysine, FBS, and / or collagen. It is also possible to treat polystyrene with sulfuric acid, and the polyurethane scaffold can be treated with fibronectin, for example, as described in section 6.4 of the Examples below.

For in vivo implantation of three-dimensional cultures, for example, biodegradation such as polyglycolic acid, collagen, collagen sponge, collagen fabric, intestinal suture material, gelatin, polylactic acid or polyglycolic acid and copolymers thereof It may be preferable to use a sex matrix. Non-biodegradable materials such as nylon, dacron, polystyrene, polyacrylate, polyvinyl, polyurethane, teflon, cotton and the like may be preferred if the culture is to be kept for a long period or stored frozen.

According to a preferred embodiment of the present invention, a helical or other concentric design that assists the cells to be aligned circumferentially under shear stress when the tubular macrostructure is formed is microstructured. The cells are inoculated on or in a biodegradable or bioresorbable scaffold having therein. Also, the scaffolding material is biodegradable, which can aid cell alignment. This is because, as the scaffold biodegrades over time, additional space is provided for cultured cells to grow inward, allowing the reconstitution of the cultured tissue under shear flow. According to this embodiment, the material is preferably biodegraded within weeks to months.

As mentioned above, SMCs are inoculated on the scaffold with or without other stromal cells, such as fibroblasts. Inoculating stromal cells into a three-dimensional skeleton at a high concentration, such as about 10 ⁶ -5 × 10 ⁷ cells / ml, for example, 25 × 10 ⁶ cells / cm ³ , results in the formation of three-dimensional stromal tissue in a shorter time. Will be done.

Again, when using cultured cells for in vivo transplantation or implantation, it is preferred to harvest stromal cells from the patient's own tissue.
The growth of cells in three-dimensional stromal cell cultures is controlled by proteins (e.g., collagen,
Elastic fibers, reticulated fibers), glycoproteins, glycosaminoglycans (eg, heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), cell matrices, and / or other substances Can be further enhanced by adding to the backbone or coating them on a support.

The stromal cells may be inoculated on the scaffold before or after forming the desired shape for implantation, eg, a tube, rope, or filament. After inoculation of the stromal cells, the three-dimensional scaffold must be incubated in a suitable nutrient medium. Fish
Many commercially available media, such as er media, Iscove media, McCoy media, DMEM media, etc., may be suitable for use. To maximize proliferative activity, it is important that the three-dimensional stromal cell culture be suspended or suspended in culture during the incubation period. In addition, the culture must be periodically "fed" to remove spent media, reduce the number of cells released, and add fresh media.

During the incubation period, before beginning to grow into the skeleton opening, the stromal cells
It will grow linearly along and surround the three-dimensional skeleton. Prior to inoculating the tissue-specific cells into the stromal matrix, it is preferable to grow the cells to an appropriate degree reflecting the amount of stromal cells present in the tissue in vivo.

The skeletal opening may determine whether stromal cells have spread across or packed into the opening,
Or it must be of a suitable size so that it can fill the opening. Maintaining the active growth of stromal cells that extend across the scaffold will promote the production of growth factors produced by the stromal cells, so that the culture will continue for a long time. For example, if the opening is too small, stromal cells may reach the confluent state quickly but cannot easily exit the mesh, and the trapped cells may exhibit contact inhibition, It may stop the production of the appropriate factors needed for sustained growth and continued culture over a long period of time. If the opening is too large, the stromal cells may not be able to spread across the opening, thus producing the appropriate factors needed for sustained growth and continued culture over long periods of time. Will decrease. When using a mesh type support, about 150 μm
An aperture in the range of about 220 μm will work well. However, depending on the three-dimensional structure and complexity of the skeleton, other sizes may work as well. Indeed, any shape or structure that allows stromal cell expansion and replication to persist and proliferate over a long period of time will exhibit a function compatible with the present invention.

In accordance with one embodiment of the present invention, exemplified in Section 6.4 of the Examples, a tubular polyurethane scaffold treated with fibronectin was used to inoculate stromal cells in a three-dimensional culture. For more details, see Thomson et al., Polymer Scaffold Processing.
, Principles of Tissue Engineering, RP Lanza, R. Langer and WL Chick (
Ed.), Pp. 263-272 (RG Landes Co, Austin, TX) and the solvent-cast / particulate leaching method described in US Pat. No. 5,514,378.
Was used to make a polyurethane scaffold (see Section 6.4 below). The porosity (VF) of the scaffold can vary, but the preferred range is from about 80% to about 95%. According to a preferred embodiment, the VF is 90%. The pore size of the scaffold can range from 63 to 500 μm, with a preferred range of 150 to 300 μm. As described, below, in Section 6.4, inoculated with smooth muscle cells on such a polyurethane scaffold, under shear flow stress, for example, at 0.3 dynes / cm ^2, by culturing for 3 weeks, the The cells aligned perpendicular to the direction of the shear flow stress, ie, similar to their in vivo orientation in blood vessels.

By supplying different types of collagen in various ratios on a support, tissue-specific cells or cells that can later be inoculated on stromal tissue or grown on structures in vivo. It can affect the growth of other cells. The proportion of collagen types supplied can be manipulated or increased by selecting stromal cells that produce the appropriate collagen type and inoculating such stromal cells onto the scaffold. For example, fibroblasts can be selected using monoclonal antibodies of the appropriate isotype or subclass that can activate complement and define a particular collagen type. By using these antibodies and complement, negative selection can be performed on fibroblasts that express the desired collagen type. Alternatively, the stroma used to inoculate the matrix may be a mixture of cells that synthesize the desired appropriate collagen type.
The distribution and origin of the five types of collagen are shown in Table I.

[0071]

[Table 1] Thus, depending on the tissue to be cultured and the desired collagen type, it is possible to select the appropriate stromal cells to inoculate the three-dimensional skeleton.

Similarly, the relative amount of collagenous and elastic fibers present in the stromal layer can be controlled by controlling the ratio of collagen producing cells to elastin producing cells in the initial inoculum.
Can be adjusted. For example, since the inner wall of the artery is rich in elastin, the arterial stroma should contain high concentrations of undifferentiated smooth muscle cells that produce elastin.

During incubation of the three-dimensional stromal cell culture, proliferating cells may be released from the matrix. These released cells can attach to the walls of the culture vessel, and the attached cells can continue to grow and form a confluent monolayer. This needs to be prevented or minimized, for example, by removing released cells at the time of supply, or by transferring the three-dimensional stromal culture to a new culture vessel. The presence of a confluent monolayer in the container will "stop" the growth of cells in the three-dimensional matrix and / or culture.
Removal of the confluent monolayer or transfer of the culture to fresh medium in a new container will restore the growth activity of the three-dimensional culture system. Such removal or transfer must be performed in any culture vessel having a stromal monolayer greater than 25% confluent. Alternatively, the culture system may be agitated to prevent the released cells from attaching, or instead of supplying the culture periodically, fresh medium may be continuously passed through the system. The culture system may be set to flow. The flow velocity is
Adjustments can be made to maximize growth in the three-dimensional culture and to wash away cells released from the culture, so that the released cells are in a container. Will not grow until it has adhered to the walls and becomes confluent.

The live stromal tissue so formed can be used as a modifying structure in vivo. Alternatively, other cells, such as tissue-specific cells or parenchymal cells, may be inoculated and grown on living three-dimensional stromal tissue and subjected to in vivo implantation.

After the three-dimensional stromal cell culture has reached the appropriate degree of proliferation, for example, additional cells such as tissue-specific cells or surface layer cells that are desired to be cultured are further placed on living stromal tissue. May be inoculated. Such cells inoculated on living stromal tissue,
It is incubated to anchor cells to stromal tissue and can be implanted in vivo, but can continue to grow where it is implanted. Alternatively, cells can be grown on living stromal tissue prior to implantation in vivo to form a culture comparable to native tissue. A higher concentration of cells in the inoculum will advantageously increase growth in the medium much more quickly than at a lower concentration.

[0076] The cells selected for inoculation will depend on the tissue to be cultured. in this case,
According to the present invention, SMC and / or endothelial cells derived from blood vessels may be selected. Thus, in one embodiment, the living stromal matrix described above may be supplemented with SMC and / or endothelial cells as tissue-specific cells.

The SMC and / or endothelial cells used in the tissue-specific inoculum were as described in Section 5.1.1 above.
It can be obtained from a cell suspension by degrading the desired tissue using standard techniques as described for obtaining stromal cells in section. For a review of methods available for obtaining parenchymal cells from various tissues, see the previous section.
See Freshney, Ch. 20, pp. 257-288.

As mentioned above, it is not necessary to add growth and regulatory factors to the medium. This is because these types of factors are produced by three-dimensional stromal cells. However, by adding such factors or inoculating other specific cells, it is possible to promote, modify, or regulate growth and cell maturation in the medium. Growth and activity of cells in culture are altered by various growth factors such as insulin, growth hormone, somatomedin, colony stimulating factor, erythropoietin, epidermal growth factor, liver erythropoiesis factor (hepatopoietin), and hepatocyte growth factor be able to. Other factors that regulate growth and / or differentiation include prostaglandins, interleukins, and naturally occurring caron.

5.2 Application of Shear Flow Stress to the Constructs of the Invention According to the invention, monolayer or three-dimensional cultures as described above are subjected to a steady laminar shear flow stress. When such stress is applied to the culture, the SMC aligns perpendicular to the direction of flow, resulting in a more similar cell alignment to that of cells in the body.

The shear flow stress may be steady and laminar, and its level may range from about 0.1 to
About 100 dynes / cm ^2, preferably about 0.3 to about 50 dynes / cm ^2, more preferably from about 0.3
Values can range from about 25 dynes / cm ² . According to one embodiment, for example, shear flow stress is at least about 15 dynes / cm ² level, more preferably in the level of at least about 20 dynes / cm ^2, and the duration of at least 24 hours, more preferably Added over a duration of at least 48 hours. According to another embodiment, the shear flow stress is applied at a level of about 0.3 dynes / cm ² for a duration of about 3 weeks. One of skill in the art can readily determine the appropriate level of shear flow stress to be applied to align within the desired time by empirical methods.

[0081] Shear flow stress can be applied to cultured cells by various means. For example,
Shear flow stress can be applied by growing an SMC monolayer on the surface of a rotating drum or rotating disk immersed in a liquid growth medium (see, eg, FIGS. 2 and 3). Alternatively, shear flow stress can be applied by growing the SMC monolayer on a stationary parallel plate through which the pumped liquid growth medium passes (see, eg, FIG. 4). The SMC monolayer was subjected to a shear flow stress of 20 dynes / cm ² in a parallel plate system as described in Section 6.2 of the Examples below. Subjecting the cells to this shear flow stress for 48 hours induced SMC alignment perpendicular to the direction of flow. Furthermore, increasing the shear flow stress induced significantly more vertical cell alignment compared to static cultures.

Shear flow stress can also be applied to a three-dimensional SMC culture by placing the culture in a growth chamber through which a liquid, such as a pumped growth medium, passes (see FIG. 1). .

The magnitude of the shear flow stress applied to the culture is controlled by adjusting the rotation speed of the drum or disc, or by adjusting the pumping rate. The shear flow stress when using a parallel plate chamber for monolayer culture is
Calculated according to (1). Shear flow stress = τ = 6 μQ / bh ² dynes / cm ² where μ = viscosity of liquid (Nsec / m ² ) Q = flow rate (ml / min) b = width of chamber (cm), and h = chamber In the culture of a three-dimensional tube, the following equation (2) is used. τ = 32μQ / πd ³ d = tube diameter (in cm) μ = dyne × sec / m ² Q = cm ³ / sec

An example of a bioreactor flow system that can be used in the method of the present invention is shown in FIG. As shown in FIG. 7, a circulating flow system 16 is used with the growth chamber 11. The circulating fluid system 16 includes a medium reservoir 9, a pump 10, a growth chamber 11, and a tube 12.

For use as reservoir 9, any sterilizable liquid container is available. One type of preferred reservoir is a sterile bag. Suitable sterile bags are commercially available, for example, from Gibco / BRL. In some embodiments of the present invention, the upper reservoir can be located upstream of the bioreactor, the lower reservoir can be located downstream of the bioreactor, and a pump, such as a liquid medium, is placed on the lower side of the bioreactor. The reservoir can be returned to the upper reservoir.

To provide a direct supply of sterile gas to the liquid in the system, the reservoir 9 can be equipped with a sterile filter. Alternatively, the reservoir 9 may include a gas permeable tube or membrane made of silicone, Teflon, or the like, to provide an indirect supply of sterilizing gas to the system by diffusion. Preferably, one or more valves and one flow meter are disposed in the flow system.

The pump 10 is designed to move liquid from the reservoir 9 to the growth chamber 11 and recirculate the liquid under sterile conditions. Typically, pump 10 controls both the flow rate and the pressure in the system. Pump 10 may be a peristaltic pump. Alternatively, an elastic bladder may be used with the fluctuating pressure source. Changing the external pressure causes the bag to bulge or dent. By using a pair of check valves, the sterile fluid can be moved in a single direction in the system. The connecting tube 12 for circulating the sterile liquid in the system may be a stainless steel pipe or a durable medical grade plastic tube. Alternatively, tube 12 may be a gas permeable material such as silicone.

The flow system of FIG. 7 can be used for culturing either a monolayer cell culture or a three-dimensional cell culture. In this case, the three-dimensional cell culture can be placed in a growth chamber and shear flow stress can be applied by the movement of a liquid pumped through the growth chamber. Preferably, the three-dimensional scaffold containing the attached cultured cells is stationary.

Other shear flow systems that can be used include those shown in FIGS. The system shown in FIG. 8 comprises a growth chamber 3 containing concentric drums 1 and 2. The surface of one or both drums can serve as a substrate for attaching cells according to the present invention. For example, SMC can be inoculated on a drum surface to form an SMC monolayer, and the monolayer can be subjected to shear flow stress. With one drum rotated, the other drum can be held stationary. In either arrangement, the drums 1, 2 are at least partially immersed in a liquid, such as a growth medium 15. In addition, both drums can be rotated. Shear flow stress is generated by the relative movement of the liquid 15 and the cells fixed to the drum.

The magnitude of the flow stress can be adjusted by adjusting the rotation speed of the drum according to the above equation (1). The distance between the drums 1 and 2 is a parameter h in the equation (1). Preferably, the rotational speed of the drum is selected so as to obtain the shear flow stress ranges listed above. Preferably, the drum is rotated using a variable speed electric motor. The optimum distance between the two drums depends on various factors, including the size of the drum and the material from which the drum is made. Determining the optimal drum configuration is within the ability of those skilled in the art. Since the shear flow stress is generated by the rotation of the drum, a continuous flow arrangement with a liquid reservoir and a pump is optimal.

The shear flow system of FIG. 9 includes a growth chamber 5 with a rotating disk 4. The disk 4 is immersed in a liquid 15 such as a culture medium. In this case, the disc functions as a substrate for attaching cells. For example, an SMC can be inoculated on a disk surface to form an SMC monolayer, and the monolayer can be subjected to shear flow stress. The magnitude of the flow stress applied to the cells can be adjusted by adjusting the rotation speed of the disk according to the above-described equation (1). Optionally, multiple disks can be placed on a single rotating shaft to increase the total surface area available to sustain cell growth. Typically, the drum is rotated using a variable speed electric motor. Since the shear flow stress is generated by the rotation of the drum, a continuous flow arrangement with a liquid reservoir and a pump is optimal.

The parallel plate flow system shown in FIG. 10 includes a shear flow growth chamber 8 with two parallel stationary plates or walls 6 and 7 inside the growth chamber 8.
In the same growth chamber 8, a pair of stationary plates 6, 7 can be used, or two or more pairs of plates can be used. Since the plates 6, 7 are stationary, the shear flow stress is
It occurs only by the movement of a liquid 15, such as a medium, pumped through the chamber 8. The liquid is pumped through the parallel plates to obtain the desired shear flow stress. As mentioned above, the shear flow stress may range from about 0.1 to about 100 dynes / cm, or more preferably, the shear stress is at least about 15 to about 20 dynes.
/ cm2 level. The shear flow stress can be adjusted by adjusting the flow rate of the liquid 15 according to the above equation (1). The distance 13 between the stationary plates 6 and 7 is a parameter h in the equation (1). The stationary plates are preferably parallel to each other and perpendicular to the main flow of the liquid 15.

An advantage of increasing the number of plates 6, 7 is that the total surface area on which cells can form a monolayer can be increased. A disadvantage that can be caused by multiple plates is that as the number of plates increases, it can be relatively more difficult to maintain a uniform level of shear flow stress on the cells throughout the chamber 8. It is.
One way to maintain a uniform level of shear flow stress is to distribute the incoming liquid 15a over a large area on one wall of the chamber 8 while simultaneously outflowing the liquid 15b.
From a similarly large area on the opposite wall of the chamber.

In some cases, the shear flow stress device utilized must be capable of changing or adjusting the flow rate, pH, and / or temperature of the medium. The device may also include image capture / processing means to evaluate alignment. The drums, disks, and plates described above must be made from a biocompatible material, such as, for example, polystyrene, polycarbonate, or stainless steel. Substrates can also be made from non-porous biodegradable materials such as scaffolds, provided that these substrates limit growth to monolayer culture. The selection of suitable materials for the walls of the growth chamber and the substrate is within the ability of those skilled in the art.

5.3 Alignment of SMCs under Shear Flow Stress Applying a shear flow stress as described above to an SMC culture described herein,
The alignment of the cells changes with the direction of the shear flow stress so that the SMC aligns perpendicular to the direction of flow. See, for example, Sections 6.2, 6.3, and 6.4 of the examples described below. As shown in FIG. 1, the alignment of dog vascular SMCs grown in static culture is essentially random. In contrast, when the cultured cells were subjected to a shear flow stress of 20 dynes / cm2 for 48 hours, the cells were aligned perpendicular to the direction of flow. Vertical alignment was dependent on the magnitude (FIG. 2) and duration (FIG. 3) of the shear flow stress exposure. The vertically aligned SMC cultures according to the invention can be used in vivo.
A more similar cell alignment to that of the cells in E. coli allows the creation of improved SMC tissue engineering constructs for in vivo implantation.

The alignment of the SMCs can be performed using any method known to those skilled in the art, for example, but not limited to, by visual observation, or by a microscope or other such as a phase contrast or fluorescence imager. Visualization is possible using an optical imaging device. Cells may be stained or immunostained to aid visualization.

5.4 Use of the Methods and Constructs of the Invention 5.4.1. Formation of Tubular Biological Tissues The three-dimensional cultures and constructs of the present invention grown under shear flow stress can be used in vivo to produce single-layer or multi-layer tubular tissues. Can be used to construct Circumferentially (
That is, these tubular structures having aligned smooth muscle tissue with or without longitudinally oriented (ie, parallel to the direction of flow) endothelial cells, i.e., perpendicular to the direction of flow, can be used in vivo. Will be much more similar to vascular tissues, organs, such as, but not limited to, blood vessels, ligaments, and tendons. Accordingly, these structures can be used as improved implantable implants and replacement structures for repairing damaged tissue in vivo.

[0098] Tubular structures, such as blood vessels, are composed of multiple layers of interstitial tissue with an inner layer of endothelium. These connective tissues contain varying degrees of elastic fibers and multiple layers of smooth muscle, all of which are particularly prominent in arterial blood vessels. In addition, such structures often contain fibroblasts and varying amounts of collagen. By applying shear flow stress to the various components of such vessels, for example, cells, including SMCs, endothelial cells, fibroblasts, etc., the method of the present invention further enhances the tissue components found in vivo. A three-dimensional implantable tissue construct or structure having reflected components is provided. Thus, these implantable structures can acquire specific structural and functional properties required to perform appropriate physiological functions in vivo. Thus, they can function as improved replacements for damaged or diseased tubular tissue in vivo.

5.4.1a. Single-Layer Tubular Structure In the following segment, we will prepare a tubular structure that can be implanted in vivo using various scaffold scaffolds for growing cells under shear flow stress. Is explained.

(A) Planar mesh starting material The scaffold can be cut into rectangular strips having a width approximately equal to the inner circumference of the tubular organ to be finally inserted. The scaffold can be inoculated with SMC and / or other stromal cells and incubated in a liquid medium under shear flow stress according to the invention. At the stage of proper confluence, the long edges can be joined and the scaffold can be rolled into a tube to form an improved properly aligned tubular implantable tissue construct. The seam can be closed by suturing the two edges using fibers of a suitable material having the appropriate diameter.

(B) Tubular Scaffold Starting Material Alternatively, initially forming the scaffold as a tube, inoculating SMC and / or other stromal cells, and growing under shear flow stress in culture media according to the present invention Can be. The tubular structure is secured by securing one of the open ends of the tubular scaffold to a nozzle and extruding the liquid medium through this nozzle from a source chamber connected to the incubation chamber so that the cells do not block the lumen. A flow through the interior of the object can be formed. The other open end can be fixed to an outflow aperture that is connected to a collection chamber that can recirculate the medium through the source chamber. When the incubation is over, the tube can be removed from the nozzle and outflow aperture. For information on how to do this, see Baller
mann, BJ et al. in International Application No.WO 94/25584 (U.S. Pat.
See also Issue 3). The improved, properly aligned tubular structures thus produced can be implanted in vivo.

(C) Multilayer Tubular Structure In another embodiment, at least two three-dimensional cultures grown under shear flow stress can be combined into a multilayer tubular structure by various methods. For example, forming a multilayer planar scaffold by placing one planar rectangular culture on another and suturing the two together or annealing them together using a photoactivator. it can. The second culture was then described as above for the first culture, i.e., by joining the long edges and suturing with suture.
The layer sheet can be rolled into a tubular structure. Alternatively, one of the tubular scaffolds to serve as the inner layer is inoculated and incubated under shear flow stress, and the second scaffold is also subjected to a flat surface having a width slightly larger than the outer circumference of the tubular scaffold, also under shear flow stress. They can be grown as rectangular strips. After reaching the appropriate degree of growth, a rectangular scaffold can be wrapped around the periphery of the tubular scaffold. The seam of the outer strip can be closed and fixed to the inner tube with a single stitching or annealing step,
The result is a multilayer, tubular, implantable structure.

In addition, two tubular meshes having slightly different diameters can be grown separately under shear flow stress. Cultures with smaller diameters can be inserted inside the larger culture and sutured with sutures. This method may not be practical for very thin tubes.

In any of these methods, more layers can be added by repeating the method on the bilayer. Further, by utilizing any suitable method, a three-dimensional culture can be shaped to take on the composition of the natural organ or tissue that mimics it.

(D) Arterial and Vein Tubular Structures Preferred embodiments of the multilayer tubular structures of the present invention include arterial and venous tubular structures . As mentioned earlier, blood vessels are composed of three layers: the intima, the media and the adventitia, in order from the inside to the outside. The major cell component of the intima is endothelial cells, and the major cell component of the media is smooth muscle cells that produce much of the extracellular matrix of vascular tissue (eg, proteins such as elastin). In the case of arteries, the inner elastic membrane, which is inside the intima and outside the media, is a homogeneous layer of fenestrated elastin. The richness of elastin in their walls allows arteries to stretch with each contraction of the heart. The veins also have elastin heterogeneous fibers or individual fibers in their walls. The adventitia is composed primarily of fibroblasts, the more common vessels (vasa vasorum), and some neural tissue. The adventitia of arteries rather than veins also has a homogeneous, fenestrated outer elastic membrane. Collagen and other individual elastins in this layer are important in anchoring blood vessels. Also, veins are tubular structures lined with endothelial cells, but the connective tissue layers are less well-defined than those of arteries and the walls are thinner. Also, human veins have poor internal and external elastic membranes.

There are compositional and functional differences in any of the layers of blood vessels. For this reason,
In accordance with the present invention, it may be advantageous to grow the various layers on separate scaffolds. Whether the intima and media are grown on separate scaffolds or combined will depend on how these layers differ in the particular artery in which the three-dimensional culture is implanted.

For example, according to the present invention, fibroblasts are isolated from the adventitia of a patient's artery and used to create a three-dimensional skeleton as described in Section 5.1.3. Above. It can be inoculated and grown according to the invention under shear flow stress. Cells are isolated from tissues that are rich in elastin-producing undifferentiated smooth muscle cells and also contain some fibroblasts (eg, the intima and media of the same artery) and are used to separate scaffolds or the same. (Ie, as tissue-specific cells on a living stromal matrix as described in Section 5.1 above). In each case, two scaffolds or a single three-dimensional culture are grown under shear flow stress. After growing the SMCs to an appropriate degree and aligning them perpendicularly to the direction of flow, tubular structures can be formed by combining the scaffolds using one of the methods described above (separate When using a scaffold). In this way, an improved arterial graft having a connective tissue layer similar to that found in arteries in vivo can be produced.

To create an arterial graft that more closely resembles the artery found in vivo, the endothelial cells were isolated from the same patient (or other source as described above) and the vertically aligned SMC Can be inoculated on culture (e.g., as described above)
. For example, endothelial cells can be seeded as tissue-specific cells on a living stromal matrix containing SMCs, as described in Section 5.1.3. Above.
The interstitial matrix was previously subjected to a shear flow stress, aligning the SMC perpendicular to the direction of flow. After inoculation of the endothelial cells onto the matrix, the culture is grown again under shear flow stress and the endothelial cells are arranged parallel to the direction of flow.
Next, when the scaffold is shaped into a tubular structure as described above, an implantable graft very similar to an in vivo artery, i.e., an endothelial lining having cells aligned parallel to the direction of flow, An implant is obtained containing the lower SMC layer with cells aligned perpendicular to the direction of flow (see also section 6.4 in the Examples below).

If a complete functional replacement with all the various layers of tissue is not required, a simple, homogeneous, three-dimensional elastin-rich, eg, SMC-containing, stromal culture may be subjected to shear flow stress. It can be propagated and used. Combining other layers of this homogeneous interstitial matrix can provide a suitable thickness for such a prosthesis.

The layers of connective tissue that make up the walls of the veins are less defined than those of the arteries.
Thus, a single three-dimensional culture can be grown, for example, from a single inoculum of cells. By isolating these cells, consisting primarily of fibroblasts and having some smooth muscle cells, from the wall of the patient's vein and growing them under shear flow stress as described herein Cultures can be made with SMCs aligned perpendicular to the direction of flow. This culture can also be formed into a tubular structure, as described above. Alternatively, when the stromal culture has reached an appropriate confluency, for example, inoculating endothelial cells isolated from the same patient onto the stromal layer,
It can be grown to confluency under shear flow stress. It is then also possible to form the culture into a tubular structure, whereby an inner endothelial layer oriented parallel to the direction of flow and two or more inner SMCs oriented perpendicular to the direction of flow. A venous structure with layers will be obtained. As mentioned earlier, these preferred embodiments best reflect the natural state of the blood vessels, and therefore the mechanical and hemodynamic stresses that would be experienced when implanted in vivo. Most likely to endure.

5.4.2. Implantation / Implantation The tissue engineered constructs described herein can be implanted in vivo to correct a defect or replace surgically excised or damaged tissue. Can be. Implantation can be performed using either monolayer or three-dimensional cultures, as described herein. The construct is preferably implanted to repair or replace structures containing smooth muscle cells and the SMC cell layer, such as blood vessels, for example, small, medium, or large tubes. Preferred implantable structures include tubular arterial and venous grafts.

In addition, the constructs are useful for repairing or replacing involuntary visceral smooth muscle, such as found in the stomach, intestine, uterus, or bladder. The constructs can also be used to repair or replace ligaments, tendons, and chordae (tricuspid and mitral tendon chords of the heart). Ligaments or tendons (including fibroblasts and collagen fibers)
In this case, as described herein, a stromal monolayer culture or a three-dimensional stromal living matrix or three-dimensional SMC culture can be used.

5.4.3. Genetic Engineering and Gene Therapy Monolayer and three-dimensional constructs grown under shear flow stress as described herein assist in implantation / implantation or result. Vehicles for introducing genes and gene products to improve and / or for use in gene therapy may be provided. According to a preferred embodiment, a three-dimensional construct is used. For example, in the case of a vascular graft, to express an anticoagulant gene product to reduce the risk of thromboembolism, or to express an anti-inflammatory gene product to reduce the risk of insufficiency due to an inflammatory response. The stromal or tissue-specific cells of the construct, eg, SMC, endothelial cells, or fibroblasts, can be engineered. In this regard, cells can be engineered to express, for example, TPA, streptokinase, or urokinase to reduce risks such as coagulation. In the case of a vascular or other type of tissue graft, additionally or alternatively, a peptide corresponding to the idiotype of an anti-inflammatory gene product, e.g., a neutralizing antibody to TNF, IL-2, or other inflammatory cytokine Alternatively, cells can be engineered to express a polypeptide. In addition, a gene encoding a human complement regulatory protein that prevents graft rejection by the host may be inserted into human fibroblasts. McCurry et al., 1995
, Nature Medicine 1: 423-427. Preferably, under transient and / or inducible control during the post-operative recovery period, or as a chimeric fusion protein anchored to the cell, for example, the intracellular domain of a receptor or receptor-like molecule and / or The cells are genetically engineered to express such a gene product as a chimeric molecule consisting of a transmembrane domain and fused to the gene product as an extracellular domain.

In another embodiment, the stromal or tissue-specific cells of the construct are expressed so as to express a gene that is deleted or has a therapeutic effect in the patient, eg, HDL, apolipoprotein E, and the like. It can be processed by genetic engineering. The gene to be introduced into cells by genetic engineering must be associated with the disease to be treated. For example, in the case of a vascular disease, the cells can be engineered to express a gene product carried by the blood, such as cerebredase, adenosine deaminase, α-1-antitrypsin. In certain embodiments, gene products such as α-1-antitrypsin can be delivered to the lung by using a genetically engineered vascular graft culture that is implanted to replace a portion of a vein or artery. . In such an approach, constitutive expression of the gene product is preferred. According to another embodiment, wound healing at the site of implantation is incorporated by incorporating genetically engineered cells expressing a wound healing factor into living stromal culture used to make tendons and ligaments. It is possible to promote.

In addition, genes that prevent or reduce the symptoms of various types of vascular disease can be under-expressed or down-regulated in disease conditions. In particular, the expression of genes involved in the prevention of the following pathologies may be down-regulated:
The following pathologies are, for example, thrombus formation, inflammatory response, and fibrosis, and calcification to valves. In this case, by introducing the expression of the gene encoding the integrin antagonist into cells, it can be used for treating abnormal cell migration. Similarly, cells can be treated to include a gene for a IIb / IIIa blocker for treating thrombosis, for example. For example, Nikol et al.
, 1996, Atherosclerosis 123: 17-31.

[0116] In addition, reduced activity of the gene product may manifest some or all of the above conditions and consequently progress to valvular disease symptoms. In this case, increasing the activity level of the gene, either by increasing the level of the gene product present in the monolayer or three-dimensional culture system, or by increasing the level of the active gene product present there Is possible. The culture expressing the active target gene product can then be implanted into a valvular disease patient deficient in that product. "Target gene", as used herein, is limited in that modulation of the level of expression of the target gene or the level of activity of the target gene product can act to reduce the symptoms of the disease. But not genes involved in the disease to be treated, such as vascular disease.

The stromal cells or tissue-specific cells used in the three-dimensional culture system of the present invention include:
The expression of factors or surface antigens that promote coagulation or rejection at the site of implantation can be engineered to "knock out" the expression. The negative regulation method for reducing the expression level of the target gene or the activity level of the target gene product is described below. "Negative modulation", as used herein, refers to a decrease in the level and / or activity of a target gene product relative to the level and / or activity of the target gene product when no regulatory treatment is applied. Means
Expression of genes specific to stromal cells and the like can be reduced or knocked out using several techniques. For example, expression can be suppressed by completely inactivating the gene using a homologous recombination method (generally, ``
Knockout "). Usually, the presence of a positive selectable marker (e.g., neo) in an exon encoding a critical region of a protein (or an exon 5 'to that region) inhibits the production of normal mRNA derived from the target gene, as a result,
The gene is inactivated. It is also possible to inactivate a gene by making a deletion in a part of the gene or deleting the entire gene. By using a construct having two regions having homology to the target gene at distant positions in the genome, a sequence flanked by the two regions can be deleted. Mombaerts, P., et al., 1991, Proc. Nat. Acad. Sci. USA 88: 3084-
3087.

[0118] Antisense and ribozyme molecules that suppress the expression of the target gene can also be used in accordance with the present invention to reduce the level of activity of the target gene. For example, antisense RNA molecules that suppress the expression of the major histocompatibility complex (HLA) have proven to be the most versatile with respect to the immune response. Furthermore, triple helix molecules can be used to reduce the level of activity of the target gene. These techniques are described in LG Davis, et al., Eds, Basic Methods in Molecular Biol.
ogy, 2nd ed., Appleton & Lange, Norwalk, Conn. 1994.

In another alternative, the cells of the construct are genetically engineered to proliferate and migrate smooth muscle cells, for example, by antisense oligodeoxynucleotide inhibition of expression of cell division cycle 2 kinase and proliferating cell nuclear antigen. , And can block gene expression required for transition to neointimal hyperplasia. Mann, MJ
, et al., 1995, Proc. Natl. Acad. Sci. USA 92: 4502-4506.

Using any of the above techniques, the expression of fibrinogen, von Willebrand factor, factor V, or any cell surface molecule that binds to the α2Bβ-3 receptor of platelets is knocked out in stromal cells. However, the risk of clump formation in blood vessels or other types of living tissue grafts can be reduced. Similarly, MHC class II
Expression of the molecule can be knocked out to reduce the risk of graft rejection.

[0121] Methods that may be useful for genetically engineering cells of the invention are well known in the art and are further described in US Pat. Nos. 4,963,489 and 5,785,964. The disclosures of these patents are incorporated herein by reference. For example, a recombinant DNA containing an exogenous nucleic acid encoding a gene product of interest, etc.
Constructs can be constructed and used to transform or transfect stromal or tissue-specific cells described herein. Transformed or transfected cells having the exogenous nucleic acid and capable of expressing the nucleic acid in the form of a gene product are selected and clonal propagated in the three-dimensional culture system of the present invention.

Methods for preparing DNA constructs containing the gene of interest, transforming or transfecting cells, and selecting cells having and expressing the gene of interest are well known in the art. It is. For example, Maniatis et al.,
1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Labora
tory Press, Cold Spring Harbor, NY; Ausubel et al., 1989, Current Protoc
ols in Molecular Biology, Greene Publishing Associates & Wiley Interscie
nce, NY; and Sambrook et al., 1989, Molecular Cloning, A Laboratory Ma.
^{nual, 2 nd ed., Cold} Spring Harbor Laboratory Press, Cold Spring Harbor,
See the technique described in NY.

For example, the cells of the invention can be transformed using a DNA construct. That is, it can be controlled by appropriate expression control elements (eg, promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selection marker. Following the introduction of exogenous or exogenous DNA, the treated cells can be grown in enriched media and then transferred to selective media. The selectable marker in the recombinant plasmid confers selectivity and also allows cells to take up the plasmid into their chromosomes and grow to form foci. This foci is then cloned and propagated into a cell line. This method can be used advantageously to form a cell line that expresses the gene protein product.

Three-dimensional cultures containing such genetically engineered cells, for example, a mixture of cells each expressing a different desired gene product or cells that have been treated to express several specific genes An object is implanted in a patient to reduce the symptoms of a disease such as, but not limited to, a vascular disease. Gene expression may be under the control of a non-inducible (ie, constitutive) promoter or an inducible promoter. The level of gene expression and the type of gene regulated can be controlled depending on the type of treatment applied to an individual patient.

A variety of methods can be used to effect constitutive or transient expression of a gene product to be introduced into a tissue-assembling cell. For example, Seldon, RF, et al
, 1987, Science 236: 714-718, the transkaryotic embedding method can be used. By "nuclear modification" as used herein is meant that the nucleus of the implanted cell has been modified by the addition of a DNA sequence by stable or transient transfection. Cells can be engineered using any of a variety of vectors. Such vectors include integrating viral vectors, such as retroviral vectors or adeno-associated viral vectors, or non-integrating replicating vectors, such as papilloma viral vectors, SV40 vectors, adenoviral vectors; or replication-defective viral vectors, and the like. But not limited thereto. If transient expression is desired, non-integrative vectors and replication deficient vectors are preferred because these systems can control the expression of the gene of interest using either an inducible or constitutive promoter. . Alternatively, transient expression can be performed using an integratable vector, provided that the gene of interest can be controlled by an inducible promoter.

Preferably, the expression control elements used should allow for the regulated expression of the gene such that the product is synthesized only when needed in vivo. The promoter selected will depend, in part, on the tissue and cell type being cultured. In the case of vascular grafts, genetic engineering of SMCs, endothelial cells, or other stromal cells allows gene products (delivered locally or systemically) to function in blood vessels or other physiological functions. Utilizing a shear stress promoter or other shear stress control element to produce a gene product under shear stress to help maintain or regulate, for example, prevent thrombosis, and even produce insulin at a certain level be able to. For example, by using a vascular graft that performs constitutive expression of insulin under shear flow stress, liver transplantation can be facilitated,
Or treat an insulin-related disorder, such as diabetes. Cells can also be engineered to produce the growth factor VEGF under the control of a shear stress responsive promoter. One such promoter can be derived from a gene encoding thrombomodulin, a protein that is transiently upregulated when subjected to shear stress.

Although a shear stress responsive promoter is preferred, any promoter can be used to drive expression of the inserted gene. For example, viral promoters include, but are not limited to, CMV promoter / enhancer, SV40, papilloma virus, Epstein-Barr virus, elastin gene promoter, and β-globin. If transient expression is desired, preferably such a constitutive promoter is used in a non-integrative vector and / or a replication defective vector. Alternatively, an inducible promoter can be used to induce expression of the inserted gene when needed. As mentioned above, it is possible to use a shear stress inducible promoter. Other inducible promoters include, but are not limited to, metallothionein and heat shock proteins.

Among the transcription control regions that exhibit tissue specificity for connective tissue, those that have been reported and can be used include, for example, elastin or elastase I gene regulation active in pancreatic acinar cells. Region (Swit et al., 1984, Cell 38: 639-646;
Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; M
acDonald, 1987, Hepatology 7: 425-515), but is not limited thereto. Elastin deposition has been associated with specific physiological and developmental events in various tissues, including vascular grafts. For example, in developing arteries, elastin deposition appears to be linked to changes in arterial pressure and mechanical activity. The transmission mechanism that links mechanical activity to elastin expression involves cell surface receptors. As elastin-synthesizing cells attach to elastin via cell surface receptors, the synthesis of additional elastin and other matrix proteins is driven by stress or mechanical forces in tissue (eg, constant movement of constructs in bioreactors). ) May be affected.

In yet another embodiment of the present invention, it is possible to produce a biological product in high yield by using a three-dimensional culture system in vitro. For example, cells that naturally produce large amounts of a particular biological product (eg, growth factors, regulators, peptide hormones, antibodies, etc.) or host cells that have been genetically engineered to produce a foreign gene product. Can be cloned in vitro using a three-dimensional culture system. If the transformed cells release the gene product into the nutrient medium, use standard separation techniques (HPLC, column chromatography, electrophoresis, etc.) to use the spent medium or conditioned medium, to name a few. The product can be easily isolated from the medium. "Bioreactors" have been devised that utilize a flow method to supply three-dimensional cultures in vitro. Essentially, as the fresh medium passes through the three-dimensional culture, the gene product is washed out of the culture along with the cells released from the culture. Gene products are isolated from the effluent of spent or conditioned media (eg, by HPLC column chromatography, electrophoresis, etc.).

In a further embodiment of the present invention, it is possible to use a three-dimensional culture to facilitate gene transfer. For example, and without limitation, a three-dimensional culture of fibroblast stroma containing a recombinant virus expression vector is used to transfer the recombinant virus into cells that are contacted with a stromal matrix, thereby in viv
o can stimulate viral transmission in o. Three-dimensional culture systems are a way to achieve gene transfer more efficiently than current techniques for DNA transfection.

The use of the genetically engineered three-dimensional cultures of the present invention in gene therapy has many advantages. First, since the culture contains eukaryotic cells, the gene product will be appropriately expressed and processed in culture to produce the active product. Second, gene therapy is useful only when the number of transfected cells is substantially increased, resulting in clinical value, suitability, and utility. The three-dimensional cultures of the present invention allow for an increase in the number of transfected cells and expansion of the transfected cells (via cell division).

6. Example 6.1 Isolation and Culture of Vascular Smooth Muscle Cells Collagenase and elastase digestion as described, for example, in Redmond et al., 1995, In Vitro Cell Dev. Biol. 31: 601-609. By canine blood vessels
SMC was isolated from the right coronary artery. Briefly, a coronary artery was surgically removed from an anesthetized animal and an antibiotic / antimycotic solution (1 × antibiotic / antimycotic solution, Gibco,
Rinse with sterile conditions using Gaithersburg MD). The adventitia was excised from the artery using sterile forceps, scissors, or a scalpel and rinsed with a 1 × antibiotic / antimycotic solution. 1
X Trypsin EDTA (Gibco) and 0.2% collagenase IV solution in vascular media
(medial tissue) was placed and incubated at 37 ° C. on a rotator for 15 minutes. The medial tissue is minced into small pieces and 0.25 mg / ml elastase, 0.7 mg / ml collagenase I, 0.4 mg / ml soybean trypsin inhibitor (Worthingto) in PBS (phosphate buffered saline).
n Biochem), and 0.1 mg / ml BSA in calcium- and magnesium-free pre-warmed solution at 37 ° C and swirled.
Incubated at 37 ° C. for 1 hour. The supernatant was collected, and the digested tissue was rinsed with DMEM (Dulbecco's modified Eagle's medium, Gibco) supplemented with 20% FBS (fetal calf serum; Hyclone, Logan UT). The medium was removed and combined with the recovered supernatant. Cells present in the combined supernatants were collected by centrifugation at 1200 rpm for 10 minutes.
Cell pellets were resuspended in DMEM supplemented with 20% FBS and placed in growth flasks for cell culture.

[0133] 10% FBS, 2 mM L-glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), and 1
X Isolated cells were cultured in DMEM with antibiotic / antimycotic solution. 5% CO ₂
The cells were cultured at 37 ° C. in a humidified incubator with, and the isolated smooth muscle cells were confirmed by positive immunostaining for α-smooth muscle actin and calponin and negative staining for von Willebrand factor. Before analyzing
The purified SMC culture was passaged 6-8 times.

6.2 Application of Shear Flow Stress to Isolated SMC Monolayer SMC cells using a parallel plate flow chamber as described in Frangos et al., 1988, Biotech. Bioeng. 32: 1053-1060. The culture was subjected to steady laminar shear flow stress. Briefly, in this system, the flow through the flow chamber was driven by hydrostatic pressure created by the vertical distance between the upper and lower fluid reservoirs. The pressure head was kept constant by continuously pumping the medium through a flow loop system. Parallel plate systems are machined polycarbonate plates,
It had a rectangular silicone elastic gasket and a glass thruster with a cultured cell monolayer attached. The shear stress τ applied to the cell monolayer was determined according to the following equation.

During [0135] τ = 60μ / bh ² formula, Q is flow rate (cm ³ / sec), mu is the viscosity (dynes × sec / cm ^2), b is the channel inlet width, and h is the channel Height.

In this system, cultured dog SMCs (passage 6-8) were plated on fibronectin-coated glass slides (2 μg / cm ² ) as described above,
Monolayer cultures were grown to confluence. The slide was mounted in a polycarbonate chamber equipped with a gasket and subjected to a steady laminar shear stress of 20 dynes / cm ² for up to 72 hours. The recycle medium consisted of the medium described above supplemented with 50 μg / ml ascorbic acid. Photographs of the cell monolayer were taken at specified time intervals using a 35-mm camera attached to a phase contrast microscope (Zeiss). Photos were scanned for later digital image processing using automated methods to determine orientation in microscopic images (e.g., Chaudhuri et al., 1993, Pattern Recognition Letters 14 (2)
: 147-153). The local orientation was determined by examining the local intensity gradient in a small area of each image. The algorithm was executed in the C programming language using a Silicon Graphics O2 workstation (Chaudhuri described above).
et al.). ± 90 ° to the flow direction using automated image processing
The average angular distribution of cell orientation was quantified.

6.3 Results When cells were subjected to a shear flow stress of 20 dynes / cm ² for 48 hours, the SM was perpendicular to the direction of flow, as opposed to random cell orientation without flow control.
C is now aligned. Specifically, the average orientation determined by the automated image processing method described above is significantly different between flowing and non-flowing conditions, -88.4 ° vs. + 37.6 °; p <0.
001. For example, see FIG. Furthermore, when the level of shear flow stress was increased, the cells became significantly more highly aligned perpendicular to the direction of flow, as opposed to static control, with 10.7 ° vs. 32.9 °; p <0.0001. there were. For example, see FIG. As confirmed from the histogram in FIG. 2, as the applied shear flow stress increases, the distribution of cell orientation angles becomes -90 ° and +90 from the random pattern.
It changes to a distribution centered around °. This indicates that the cells are aligned perpendicular to the direction of flow. Thus, these experiments demonstrate that the application of steady laminar shear flow stress to cultured SMCs aligns the cells in an orientation perpendicular to the direction of flow.

Furthermore, by utilizing the fluorescent staining of the F-actin filament as follows,
The effect of shear stress on SMC microfilament alignment was visualized. After the experiments described above, the SMC monolayer was washed in PBS at 37 ° C. and fixed in 3.7% formaldehyde. The cells were then permeabilized with 0.1% Triton-X100 and stained with Oregon Green 488-Phalloidin (Molecular Probes, Eugene OR). Fluorescent staining of F-actin microfilaments also showed cell orientation perpendicular to the direction of flow.

Further studies were conducted to determine the effect of the duration and magnitude of shear stress on SMC cell alignment. SMC cells were grown in monolayers as described above, and shear flow stressed the cells using the device described above at various magnitudes and durations of stress. Images of the sheared cell monolayer were captured with a CCD camera and recorded on a time-lapse VTR.

The results of this study are shown in FIG. In fact, the vertical alignment rate of SMC under shear flow stress was dependent on the magnitude and duration of the stress. For example, as shown in FIG. 3, the cells were subjected to 20 dynes / cm ^2, compared to cells attached to the 15 dynes / cm ² aligned within 48 hours, more quickly, i.e. Aligned vertically within 24 hours.
Furthermore, as shown in FIG. 4, the SMC monolayer subjected to 20 dynes / cm ² began to align perpendicular to the flow within hours after applying the shear flow stress. Image processing of time-lapse video images revealed that the change in cell orientation angle decreased dramatically by 8 hours after subjecting to shear flow stress. From this,
Increased cell alignment is suggested.

Further studies confirmed that the vertical alignment of SMCs under shear flow stress was suppressed by the addition of the calcium chelator quin2-AM. 31 ° vs
14 °; p <0.02. Specifically, SMC monolayers were treated with 10 μM quin2-AM in DMSO for 1 hour. The cells were then subjected to shear flow stress using media containing 1 μM quin2-AM, and the direction and degree of cell alignment was quantified using the automated imaging method described above. In this case, an angle variation of less than 20 ° implies significant alignment. As noted above, treatment with quin2-AM suppressed shear-induced SMC alignment as compared to untreated cells. See FIGS. 5A-5B.

Further, to determine the role of microtubule tissue in SMC alignment, prior to applying shear flow stress, SMC was incubated with 3 μM nocodazole (in DMSO), a well-known inhibitor of microtubule networks.
The monolayer was treated for 24 hours. While applying shear flow stress, 3 μM nocodazole was added into the circulating medium. Nocodazole treatment was found to inhibit SMC alignment under fluid flow. 33 ° vs. 18 °; p <0.005. As demonstrated by these results shown in FIGS. 6A-6B, intracellular calcium and microtubule networks are believed to be important in controlling SMC alignment by fluid shear stress.

6.4 Shear stress alignment of cells in three-dimensional cultures Shear flow stress was also used to align smooth muscle cells in three-dimensional cultures perpendicular to the direction of flow.

According to one embodiment of the present invention, a sterilized porous tubular polymer scaffold is briefly immersed in filtered 70% ethanol to wet the polymer surface, and then
Can be soaked for 4 hours. The medium consists of DMEM with 10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1 × antibiotic / antimycotic solution. Immobilization of serum proteins on the scaffold promotes subsequent cell attachment. Next, 13ml
Each scaffold can be placed in a 15 ml conical tube containing a suspension containing 10 ⁶ vessels SMC in medium. By rotating the conical tube at 37 ° C. for 24 hours, cells can be uniformly attached over the entire scaffold.

According to another embodiment, Thomson et al., Polymer Scaffold Processin
g, Principles of Tissue Engineering, RP Lanza, R. Langer and WL Chick (e
ds.), pp. 263-272 (RG Landes Co, Austin, Tex.) was used to make porous tubular polyurethane scaffolds using the solvent casting / particle leaching method. This procedure is also described in U.S. Pat.
It is also described in 5,514,378. Briefly, polyurethane (Tecothane; T
hermedics, Inc .; Woburn, MA) with tetrahydrofuran (Sigma # T-5267; St. Lou).
is, MO) solvent and then 150-300 μm sodium chloride (NaCl) (Sigma #
S9888) The same pore size was formed by mixing the particles with a porosity of 90% (salt: polymer weight). The polymer / salt mixture was repeatedly dip cast on a 4 mm diameter rod to make a tubular structure. Next, the structure is air-dried overnight,
The deionized water was changed several times to leached the NaCl. After drying, the scaffolds were cut to a length of 6 cm, luers were attached to each end and placed in separate conical tubes for electron beam sterilization (2.0 Mrad). The sterilized scaffold was rewet in 100% and 70% ethanol and rinsed with two changes of phosphate buffered saline (Gibco # 14190-136). To prepare a surface for cell attachment, incubator (37 ° C, 5% CO ₂
, 90% relative humidity) at 10 μg / ml sterile filtered fibronectin (Sigma
The scaffold was immersed in # F-1141) overnight.

Next, the fibronectin-coated scaffold was aspirated to a semi-dry state and passaged dog coronary artery smooth muscle cells (20 × 10 ⁶ cells / ml) (as described in Section 6.1 above). (Isolated) and DMEM (Gibco # 11960-051) in a high-density suspension for about 30 seconds (about 0.5 ml or 10 × 10 ⁶ cells fill the pores of the graft). The inoculated graft was placed in an empty 15 ml conical tube, capped tightly, and spun circumferentially in the incubator for about 4 hours (about 1 rpm). 20% fetal calf serum (Hyc
lone, # SH30070.03), 3 ng / ml cupric sulfate (Sigma # C-8027), 1% antibiotic / antimycotic (Gibco # 15240-062), 1% non-essential amino acids (Gibco # 11140-050) ), 1% L-glutamine (Gibco # 25030-081), and 1% sodium pyruvate (Gibco # 11360-070)
) Were placed in DMEM containing DMEM and loosely capped for overnight incubation in an incubator.

The inoculated graft was then cultured under constant flow in a fluid flow bioreactor, for example, as described in US Pat. No. 5,792,603. Bioreactors can regulate fluid flow through the lumen of a three-dimensional structure.
Thereby, the shear stress in the lumen wall can be controlled.

Specifically, about 50 m supplemented with 50 μg / ml L-ascorbic acid (Sigma # A-4034)
The explants were placed in a 50 ml fluid flow bioreactor containing 1 liter of the above media preparation. The explants were cultured while fluid was flowing through the lumen at a fluid shear stress of about 0.3 dynes / cm ² . The ascorbic acid supplemented medium was changed weekly (every 5-7 days) for 3-5 weeks. In addition, by performing a partial medium exchange (for example, 50%) weekly,
Fresh nutrients can be provided while retaining autocrine factors (eg, growth factors) produced by the cells.

Grafts cultured as described above (herein, tissue-treated vascular grafts (TE
VG) also demonstrated good adhesion of the smooth muscle to the graft and growth on the graft. In addition, scanning electron micrographs showed that smooth muscle tissue was present on the graft with smooth muscle cells aligned perpendicular to the direction of flow (see FIG. 11). Thus, as demonstrated by this experiment, seeding smooth muscle cells on three-dimensional tubular vascular grafts, followed by culturing the cells / grafts under shear flow stress, results in an orientation perpendicular to the direction of flow. The cells are aligned with. Also,
This alignment is similar to the alignment exhibited by SMCs in vivo, eg, in native blood vessels.

In a further embodiment of the present invention, canine microvascular endothelial cells on TEVG as described above
(MVEC) and subjected to shear flow stress. Specifically, for example, Williams S
K, Jarrell BE, Kleinert LB., 1994 (Nov-Dec), J Invest Surg 7 (6): Isolate canine MVEC from canine sickle ligament fat as described in 503-17 and passive inoculation.
40,000-60,000 cells / cm ² were inoculated into the lumen of TEVG. In addition, force deposition (force
Cells can also be inoculated by the (d deposition) method. In some cases, endothelial cells can be inoculated at a density of less than 50% confluency.

After inoculation, TEVG was placed in the bioreactor as described above and spun at 1 rpm for 4-8 hours. Attach the bioreactor to a peristaltic pump, and
The medium was pumped as a steady laminar flow with a fluid shear stress of 0.1 dynes / cm ² . After 3 days, the flow was increased to about 6 dynes / cm ² (steady laminar flow) and the TEVG was cultured for a further 3 days.

In addition, human MVEC can be used in this embodiment. Like that
MVEC is a commercial product that has been passaged three times from Cascade Biologics, Inc., Portland, OR.
And contains microvascular growth supplements (MVGS, Cascade)
Can be cultured in Medium 131 (Cascade). Cells at passage 5 should be
Recovered using Syn / EDTA (Gibco, Rockville MD) and 7.5 × 10 in supplemented Medium 131 ^Five Resuspend at cells / ml and 0.7 ml can be inoculated into the lumen of each TEVG
.

[0153] The TEVG graft of this embodiment is based on the presence of MVEC and a fluorescently labeled protein that interacts with endothelial cells, such as acetylated low density lipoprotein (Molecule).
ar Probes, Eugene, OR) or antibodies to CD31 (Pharmigen, San Diego),
It is characterized by being oriented using an antigen expressed on endothelial cells, followed by scanning electron microscopy.

As shown in FIG. 12, vascular endothelial cells seeded on SVG-containing TEVG adhered well to the luminal wall of the graft under shear flow stress and were parallel to the direction of flow. A smooth endothelial lining was formed with cells aligned with the lining. About 6 dynes /
Good adhesion was obtained at a fluid shear stress of cm ² . Very low shear flow stress,
For example, control explants cultured under about 0.1 dynes / cm ² did not show endothelial cell alignment.

The alignment of endothelial cells parallel to the direction of flow in these experiments
ivo, for example, similar to the cell alignment of natural blood vessels (e.g., Ballerman
et al., WO 94/25584). Thus, the SMCs are grown under shear flow stress, aligned perpendicular to the direction of flow, and then inoculated with endothelial cells onto the culture in the direction of flow along the luminal wall of the culture. The three-dimensional cultures of the invention aligned in parallel will be very similar in structure and morphology to those found in the vasculature in vivo.

The present invention is not limited in scope by the specific embodiments described herein. These embodiments are provided as examples of individual aspects of the invention, and functionally equivalent methods and elements are within the scope of the invention. In practice,
Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are considered to be within the scope of the appended claims.

[Brief description of the drawings]

FIG. 1 shows the application of shear flow stress to canine vascular smooth muscle cells and the resulting vertical alignment of the cells to the direction of flow. FIG. 1A shows an exemplary phase contrast image of smooth muscle cells cultured in a monolayer under static conditions. FIG. 1B shows a phase contrast image of smooth muscle cells exposed to a shear flow stress of 20 dynes / cm ² for 48 hours. Arrows indicate the direction of flow.

FIG. 2 shows the alignment of vascular smooth muscle cells with respect to the magnitude of applied shear flow stress. The phase contrast image shows the indicated shear flow stress over 48 hours, ie, 0 (static conditions) to 20 dynes.
1 shows a monolayer of smooth muscle cells after a shear flow of / cm ² . Cells showed no 1 or 10 dynes / cm ² in selective cell alignment when compared to static controls, 15 and 20 dynes / cm ²
The exposed cells were aligned perpendicular to the direction of flow. The corresponding bar graphs compare cell orientation angles by image processing. The X axis ranges from -90 to +90 with respect to the direction of the liquid flow. This bar graph confirms that as the applied shear flow stress increases, the distribution of cell orientation angles changes from a random pattern to a distribution concentrated around -90 ° and + 90 °. This indicates a vertical alignment of the cells with the direction of flow.

FIG. 3 shows the alignment of vascular smooth muscle cells in a monolayer with respect to time and magnitude of shear flow stress. Bar graph showing cell orientation angle, the cells exposed to 20 dynes / cm ^2,
It is evident that the rate of reorientation in the +/- 90 ° direction to shear stress is faster (24 hours) than cells with 15 dynes / cm ² (48 hours).

FIG. 4 shows the time course of smooth muscle cell alignment under shear flow stress, shown as angular dispersion over time. Smooth muscle cells cultured in monolayer were subjected to a shear flow stress of 20 dynes / cm ² and reorientation was initiated within the indicated time from the onset of shear stress. Image processing of the time-lapse video images revealed that the angular variance of the cell orientation angle was significantly reduced by the 8-hour experiment, indicating an increase in cell alignment.

FIG. 5. Intracellular calcium is required for smooth muscle cell alignment. This figure shows a phase contrast image of dog smooth muscle cells cultured in a monolayer under the following conditions. (A) 20 dynes / cm ² at 48 hours; and (B) quin2-AM smooth muscle cells were treated with (a chelating agent of intracellular calcium), 20 dynes / cm ² at 48 hours. Treatment of cells with quin2-AM inhibits smooth muscle cell alignment. The morphology of qin2-AM treated cells due to shear stress is similar to that of static cultured cells.

FIG. 6. Disruption of the microtubule network inhibits smooth muscle cell alignment. This figure is
1 shows a phase contrast image of dog smooth muscle cells cultured in a monolayer under the following conditions. (
A) 48 hours at 20 dynes / cm ² ; and (B) smooth muscle cells treated with nocodazole (which disrupts the microtubule network) for 48 hours at 20 dynes / cm ² . Treatment of cells with nocodazole inhibits cell alignment when compared to untreated shear stressed cells. Nocodazole-treated cells have a circular morphology when compared to static cultured cells.

FIG. 7 shows a schematic of a shear flow bioreactor system with a growth chamber, pump, media reservoir, and connecting tubing.

FIG. 8 shows a schematic of a shear flow growth chamber with concentric internal and external vessels immersed at least partially in liquid medium.

FIG. 9 shows a schematic diagram of a shear flow growth chamber with a disk rotating in the growth chamber.

FIG. 10 shows a schematic of a shear flow growth chamber with static parallel plates inside the growth chamber.

FIG. 11: 90% voids and 150-300 with a shear flow of 0.3 dynes / cm ² applied for 3 weeks.
FIG. 4 shows a scanning electron micrograph of smooth muscle cells in a three-dimensional culture on a tubular polyurethane scaffold with a pore size of μm. FIG. 11A: Hematoxylin and eosin staining of three-dimensional cultures at 70 × magnification shows smooth muscle cells growing in the scaffold. FIG. 11B shows a cross section of a three-dimensional culture. FIG. 11C: Smooth muscle cells in a three-dimensional culture at 500 × magnification show cell alignment perpendicular to the direction of flow.

FIG. 12. Compared to smooth muscle cells in a three-dimensional culture on a tubular polyurethane scaffold with 90% interstitial space and 150-300 μm pore size with 6 dyne / cm ² shear flow applied for 3 days. FIG. 2 shows a scanning electron micrograph of microvascular endothelial cells grown on a tissue-treated vascular graft (TEVG) of the present invention. Endothelial cells align parallel to the direction of flow, the orientation observed in natural blood vessels in vivo.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Seltinger, Joan United States 92122 Camino Tissino, San Diego, California 4136 (72) Inventor Cladakis, Stephanie, M. United States 30339 Lake View Lane, Atlanta, Georgia 1518 F-term (reference) 4B029 AA02 AA21 BB11 CC02 DC07 4B065 AA93X AC12 BC43 CA44 4C081 AB13 BB07 BB08 CA21 CD34 DA03 DB07 DC05 EA01 EA11 EA11 EA13

Claims (50)

[Claims] 1. A method of culturing smooth muscle cells in vitro, wherein stromal cells containing smooth muscle cells are grown under shear flow stress on a substrate, and the smooth muscle cells are grown in a shear flow direction. Such a method, comprising vertically aligning. 2. A method of culturing smooth muscle cells in vitro, comprising: a) culturing stromal cells containing smooth muscle cells on a substrate; and b) applying shear flow stress to the cultured cells. Aligning the smooth muscle cells perpendicular to the direction of the shear flow. 3. The substrate is a surface that supports smooth muscle cell growth in a monolayer.
The method according to claim 1. 4. The method according to claim 1, wherein the substrate is a scaffold that supports smooth muscle cell growth in a three-dimensional culture. 5. The method of claim 4, wherein the stromal cells, including the smooth muscle cells, are cultured on a three-dimensional scaffold made of a biocompatible non-biological material having interstices bridged by the stromal cells.
The method described in. 6. The method of claim 1 or 2, wherein the stromal cells further comprise fibroblasts, endothelial cells or other loose connective tissue cells. 7. The method of claim 5, wherein the stromal cells further comprise fibroblasts, endothelial cells or other loose connective tissue cells. 8. The method of claim 1, wherein the shear flow stress is in the range of about 0.1 to about 100 dynes / cm ² . 9. The method of claim 5, wherein the shear flow stress is in a range from about 0.1 to about 100 dynes / cm ² . 10. The method of claim 1, wherein the shear flow stress is in the range of about 0.3 to about 50 dynes / cm ² . 11. The method of claim 5, wherein the shear flow stress is in a range from about 0.3 to about 50 dynes / cm ² . 12. The method of claim 1, wherein the shear flow stress is in the range of about 0.3 to about 25 dynes / cm ² . 13. The method of claim 5, wherein the shear flow stress is in a range from about 0.3 to about 25 dynes / cm ² . 14. The method of claim 5, wherein the scaffold is biodegradable. 15. The method of claim 5, wherein the scaffold is non-biodegradable. 16. The method of claim 15, wherein the scaffold is comprised of polyurethane. 17. The method of claim 16, wherein the polyurethane scaffold has about 80% to about 95% void volume. 18. The method of claim 5, wherein the scaffold has a pore size of about 150-300 μm. 19. The method according to claim 1, wherein the stromal cells are mammalian cells. 20. The method of claim 5, wherein the stromal cells are mammalian cells. 21. The method of claim 1 or 2, wherein the stromal cells contain at least one exogenous gene under the control of an expression element. 22. The expression element is induced by shear flow stress.
The method of claim 1. 23. The method of claim 5, wherein the stromal cells contain at least one exogenous gene under the control of an expression element. 24. The expression element wherein the expression element is induced by shear flow stress.
3. The method according to 3. 25. The method according to claim 1 or 2, further comprising:
A method as described above comprising seeding endothelial cells on aligned smooth muscle cells and culturing the endothelial cells under shear flow stress to align the endothelial cells parallel to the direction of shear flow. 26. The method of claim 25, wherein the endothelial cells are seeded at a confluent concentration of less than 50%. 27. The method of claim 25, wherein the endothelial cells contain at least one exogenous gene under the control of an expression element. 28. The expression element wherein the expression element is induced by shear flow stress.
7. The method according to 7. 29. The method of claim 25, wherein the stromal cells contain at least one exogenous gene under the control of an expression element. 30. The expression element is induced by shear flow stress.
9. The method according to 9. 31. The method of claim 5, further comprising inoculating the aligned smooth muscle cells with endothelial cells and culturing the endothelial cells under shear flow stress.
Such a method, comprising aligning the endothelial cells parallel to the direction of the shear flow. 32. The method of claim 31, wherein the endothelial cells are seeded at a confluent concentration of less than 50%. The method according to claim. 33. The method of claim 31, wherein the endothelial cells contain at least one exogenous gene under the control of an expression element. 34. The expression element is induced by shear flow stress.
3. The method according to 3. 35. The method of claim 31, wherein the stromal cells contain at least one exogenous gene under the control of an expression element. 36. The expression element is induced by shear flow stress.
5. The method according to 5. 37. A method of producing an improved tubular structure for in vivo implantation, comprising the steps of: converting stromal cells, including smooth muscle cells, in vitro with interstices bridged by stromal cells. Such a method comprising culturing under shear flow stress on a tubular three-dimensional scaffold of non-biological material, thereby aligning the smooth muscle cells on the scaffold in an orientation perpendicular to the direction of the shear flow. 38. The method of claim 37, further comprising inoculating endothelial cells on smooth muscle cells aligned on the tubular structure and subjecting the tubular structure to shear flow stress. Culturing and aligning the endothelial cells parallel to the direction of the shear flow. 39. A method for producing an improved tubular structure for in vivo implantation, comprising the steps of: converting stromal cells, including smooth muscle cells, in vitro with interstitial cells interstitial spaces. Culture under shear flow stress on a planar scaffold of non-biological material, thereby aligning the smooth muscle cells on the scaffold in an orientation perpendicular to the direction of the shear flow, and allowing the scaffold for in vivo implantation. Including rolling into a tubular structure,
The above method. 40. The method of claim 39, further comprising inoculating endothelial cells on smooth muscle cells aligned on the planar scaffold structure and subjecting the scaffold structure to shear flow stress. Culturing to align the endothelial cells parallel to the direction of the shear flow, and then rolling the scaffold into a tubular structure. 41. A method for producing an improved tubular structure for in vivo implantation, comprising: (a) providing a stromal cell containing a smooth muscle cell in vitro with a gap crosslinked by the stromal cell. Culturing under shear flow stress on at least first and second planar rectangular scaffolds of biocompatible non-biological material, thereby aligning the smooth muscle cells on the scaffolds in an orientation perpendicular to the direction of the shear flow. (B) laminating a first scaffold on at least a second scaffold, and (c) rolling the scaffold into a tubular structure for in vivo implantation. 42. Inoculating endothelial cells on smooth muscle cells aligned on a first scaffold, and culturing the first scaffold under shear flow stress so that the endothelial cells are parallel to the direction of shear flow. 42. The method of claim 41, further comprising rolling the scaffold into a tubular structure after aligning the scaffold. 43. A method for producing an improved tubular structure for in vivo implantation, comprising: (a) having stromal cells, including smooth muscle cells, in vitro with gaps cross-linked by stromal cells. Culturing under shear flow stress on a tubular three-dimensional scaffold made of biocompatible non-living material, whereby the smooth muscle cells are aligned on the scaffold in an orientation perpendicular to the direction of shear flow; In vitro, stromal cells, including muscle cells, are formed in vitro by at least one planar rectangle of a biocompatible non-biological material having a width slightly greater than the outer edge of the tubular scaffold and having interstices bridged by the stromal cells. Culture under shear flow stress on the scaffold, thereby aligning the smooth muscle cells on the planar scaffold in an orientation perpendicular to the direction of shear flow, and (c) wrapping the planar scaffold around the perimeter of the tubular scaffold By in vivo implantation Forming a tubular structure for the method. 44. Inoculation of endothelial cells onto smooth muscle cells aligned on a tubular structure and culturing the tubular structure under shear flow stress such that the endothelial cells are oriented in the direction of shear flow. 44. The method of claim 43, further comprising wrapping a planar scaffold around the tubular scaffold after the parallel alignment. 45. A method of making an improved tubular structure for in vivo implantation, comprising interstitial cells having different diameters and crosslinked by stromal cells in vitro, including smooth muscle cells. Culture under shear flow stress on at least two tubular scaffolds of biocompatible non-biological material with gaps, thereby aligning the smooth muscle cells on each scaffold in an orientation perpendicular to the direction of shear flow And inserting the smaller diameter scaffold into the larger diameter scaffold to form a tubular structure for in vivo implantation. 46. Inoculating endothelial cells on smooth muscle cells aligned on a smaller diameter tubular structure, and culturing the smaller diameter tubular structure under shear flow stress, 46. The method of claim 45, further comprising inserting the smaller diameter scaffold into the larger diameter scaffold after aligning the endothelial cells parallel to the direction of the shear flow. 47. A method for producing a tissue construct for repair or replacement of a structure containing a smooth muscle cell in vivo, comprising the steps of: Culturing under shear flow stress on a substrate that supports the growth of said cells, thereby aligning said smooth muscle cells perpendicular to the direction of shear flow. 48. A three-dimensional tubular tissue construct prepared in vitro, comprising smooth muscle cells on a tubular three-dimensional scaffold consisting of a biocompatible non-biological material having interstices bridged by stromal cells. Such a tissue construct, comprising stromal cells, wherein the smooth muscle cells are aligned on the perimeter of a scaffold relative to the tubular tissue construct. 49. The tissue construct of claim 48, wherein the stromal cells further comprise fibroblasts, endothelial cells or other loose connective tissue cells. 50. The tissue construct of claim 48, wherein the stromal cells contain at least one exogenous gene under the control of an expression element.