Processes for removing cells and cell debris from tissue and tissue constructs used in transplantation and tissue reconstruction
First Claim
1. A method of treating tissue having cell membranes, excised from an animal, for making a tissue-derived, implantable bioprosthesis, the method comprising:
- contacting the excised tissue with a first detergent, wherein the first detergent is an ionic detergent capable of disrupting the cell membranes; and
contacting the excised tissue with a second detergent, wherein the second detergent has a net neutral charge, wherein the first and second detergents are both in contact with the tissue at the same time.
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Accused Products
Abstract
Methods for decellularizing mammalian tissue for use in transplantation and tissue engineering. The invention includes methods for simultaneous application of an ionic detergent and a nonionic detergent for a long time period, which may exceed five days. One method utilizes SDS as the ionic detergent and Triton-X 100 as the nonionic detergent. A long rinse step follows, which may also exceed five days in length. This long duration, simultaneous extraction with two detergents produced tissue showing stress-strain curves and DSC data similar to that of fresh, unprocessed tissue. The processed tissue is largely devoid of cells, has the underlying structure essentially intact, and also shows a significantly improved inflammatory response relative to fresh tissue, even without glutaraldehyde fixation. Significantly reduced in situ calcification has also been demonstrated relative to glutaraldehyde fixed tissue. Applicants believe the ionic and non-ionic detergents may act synergistically to bind protein to the ionic detergent and may remove an ionic detergent-protein complex from the tissue using the non-ionic detergent. The present methods find one exemplary use in decellularizing porcine heart valve leaflet and wall tissue for use in transplantation.
64 Citations
71 Claims
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1. A method of treating tissue having cell membranes, excised from an animal, for making a tissue-derived, implantable bioprosthesis, the method comprising:
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contacting the excised tissue with a first detergent, wherein the first detergent is an ionic detergent capable of disrupting the cell membranes; and
contacting the excised tissue with a second detergent, wherein the second detergent has a net neutral charge, wherein the first and second detergents are both in contact with the tissue at the same time. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 69, 70, 71)
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28. The method of claim 28, in which both the first and second detergents are in contact with the tissue within about 2 hours of the tissue being excised from the animal.
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31. A method of treating a tissue construct having cell membranes, the tissue construct being provided from a tissue culture for making a tissue culture construct product, the method comprising:
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contacting the tissue construct with a first detergent, wherein the first detergent is an ionic detergent capable of disrupting the cell membranes;
contacting the tissue construct with a second detergent, wherein the second detergent has essentially a net neutral charge, wherein the first and second detergents are both in contact with the tissue at the same time for a sufficient time to solubilize at least 90 percent of the non structural protein; and
rinsing the tissue construct to remove the detergents and proteins from the tissue construct.
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32. A method of treating a tissue derived from animal or tissue construct sources, the tissue including non-structural proteins and having a thickness and a tissue thickness center, the method comprising:
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contacting the tissue construct with a first detergent, wherein the first detergent is an ionic detergent capable of disrupting the cell membranes and binding to the non-structural proteins to form a first detergent-protein complex;
contacting the tissue construct with a second detergent, wherein the second detergent has essentially a net neutral charge, wherein the first and second detergents are both in contact with the tissue at the same time for a sufficient time insudate the tissue thickness center; and
rinsing the tissue to remove most of the first and second detergents and non-structural proteins from the tissue thickness center. - View Dependent Claims (33)
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- 34. A method of sterilizing tissue, the method comprising contacting the tissue with a sterilant selected from the group consisting of Cetylpyridinium chloride (CPC), CPC derivatives, and combinations thereof.
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38. A tissue product derived from mammalian or tissue culture sources, comprising:
a tissue thickness, in which at least about 80% of the original non-structural proteins averaged across the thickness have been removed while the initial structural integrity of the tissue has not been significantly reduced. - View Dependent Claims (39, 40, 41)
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42. A tissue product derived from mammalian or tissue culture sources, comprising:
a tissue thickness, in which at least about 80% of the original non-collagen, non-elastin proteins averaged across the thickness have been removed while the initial structural integrity of the tissue has not been significantly reduced. - View Dependent Claims (43, 44, 45, 46)
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47. A tissue product derived from mammalian or tissue culture sources, comprising:
a tissue thickness, in which at least about 80% of the original nuclei have been removed when examined histologically, when averaged across the thickness, wherein the removal is determined by the absence of both original intact size nuclei and pichnotic nuclei, while the initial structural integrity of the tissue has not been significantly reduced. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 56)
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57. A mammalian tissue-derived, implantable bioprosthesis product produced by the process comprising:
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excising a piece of mammalian tissue from a mammal, the tissue including cell membranes;
washing the tissue in a wash solution comprising about 0.1 to 1.0 percent non-phosphate saline solution, about 10 mM to 30 mM chelating agent;
a protease inhibitor cocktail, an antibacterial agent, at a pH between about 7 and 8, at a temperature of between about 20 degrees C. and 30 degrees C., for a period of between 1 to 2 days, under agitation;
soaking the tissue in a hypotonic decellurlarizing solution comprising a first detergent and a second detergent, a non-phosphate saline solution, and at least one anti-bacterial agent, wherein the first detergent is ionic and present in a concentration of between about 0.1 and 0.5 wt %, wherein the second detergent is non-ionic and present in a concentration between about 0.1 and 0.5 wt percent, at a temperature of between about 20 and 40 degrees C., with agitation, wherein the first and second detergents are both present together for a time period of at least about 3 days, wherein the first detergent is capable of disrupting the mammalian cell membranes; and
rinsing the soaked tissue for a time period of about the soak period with a rinse solution comprising a non-phosphate saline solution, an antibacterial agent, a protease inhibitor, at a temperature of between about 20 and 40 degrees C. - View Dependent Claims (58, 59, 60)
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61. A method of treating tissue excised from an animal for making a tissue-derived, implantable bioprosthesis, the method comprising:
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contacting the excised tissue with a first detergent, wherein the first detergent includes sodium dodecyl sulphate; and
contacting the excised tissue with a second detergent, wherein the second detergent has essentially no net charge;
is either a non-ionic detergent or a zwitterionic detergent operating a pH to impart a net neutral charge,wherein the first and second detergents are both in contact with the tissue at the same time and for a time period of at least 3 days. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68)
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Specification