WO2025155899A1

WO2025155899A1 - Pump-free wearable 3d skin models and systems

Info

Publication number: WO2025155899A1
Application number: PCT/US2025/012165
Authority: WO
Inventors: Hasan Erbil ABACI
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2024-01-19
Filing date: 2025-01-17
Publication date: 2025-07-24
Anticipated expiration: 2026-07-19

Abstract

A reactor, comprising: a reaction chamber, the reaction chamber comprising a porous scaffold therein, the porous scaffold having an interior volume; a first medium chamber and a second medium chamber, the synthetic tissue reactor arranged so as to place the interior volume of the porous scaffold chamber into fluid communication with the first medium chamber and the second medium chamber such that motion of the synthetic tissue reactor is configured to effect movement of a fluid among the first medium chamber, the second medium chamber, and the interior volume of the porous scaffold.

Description

PUMP-FREE WEARABLE 3D SKIN MODELS AND SYSTEMS

RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of United States patent application no. 63/622,647, “Pump-Free Wearable 3D Skin Models And Systems,” filed January⁷ 19, 2024. All foregoing applications are incorporated herein by reference in their entireties for any and all purposes.

GOVERNMENT RIGHTS

[0002] This invention was made with government support under HT9425-23-1- 0487 awarded by the Medical Research and Development Command. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present disclosure relates to the field of synthetic skin.

BACKGROUND

[0004] Each year, more than one million patients are hospitalized in the U.S. for significant skin loss due to thermal and pressure injuries, chronic diabetic ulcers or genetic skin diseases. The ability to generate engineered human skin substitutes (HSSes) has provided a promising and effective therapy for these patients, and there is a long-felt need in the art for methods and devices related to forming HSS materials.

SUMMARY

[0005] In meeting the described long-felt needs, the present disclosure provides a synthetic tissue reactor, comprising: a reaction chamber having a porous scaffold therein, the porous scaffold having an interior volume; a first medium chamber and a second medium chamber, the synthetic tissue reactor arranged so as to place the interior volume of the porous scaffold into fluid communication with the first medium chamber and the second medium chamber such that motion of the synthetic tissue reactor is configured to effect movement of a fluid among the first medium chamber, the second medium chamber, and the interior volume of the porous scaffold.

[0006] Also provided is a method, comprising: forming a synthetic skin superposed on a porous scaffold disposed in a reactor, the synthetic skin comprising dermis and epidermis, the forming comprising effecting motion of the reactor so as to effect contact between the dermis and a medium.

[0007] Further provided is a method of forming a skin substitute, comprising: forming a portion of epidermis on a dermis superposed on a porous scaffold, while forming the portion of the epidermis, effecting pump-free motion of a medium in contact with the portion of dermis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

[0009] FIG. 1A provides a depiction of a porous skin scaffold according to the disclosed technology.

[0010] FIG. IB provides an alternative view of the porous skin scaffold of FIG. 1 A.

[0011] FIG. 2A provides a depiction of a synthetic tissue reactor according to the present disclosure.

[0012] FIG. 2B provides an illustration of a synthetic tissue reactor according to the present disclosure; the reactor in FIG. 2B has been made via additive manufacturing.

[0013] FIG. 2C provides an illustration of a synthetic tissue reactor according to the present disclosure.

[0014] FIG. 3A provides a bottom lid useful in connection with a synthetic tissue reactors of the present disclosure.

[0015] FIG. 3B illustrates several synthetic tissue reactors according to the present disclosure, which reactors have been coated with an example coating that includes 8% gelatin crosslinked with 5 units/g transglutaminase. [0016] FIG. 3C illustrates a synthetic tissue reactor of FIG. 3B engaged with a bottom lid of FIG. 3 A.

[0017] FIG. 4 illustrates the synthetic tissue reactor of FIG. 3C after gelation of a mixture of Collagen Type I gel and dermal fibroblasts, after the bottom lid has been removed, and after formation and initial remodeling of the dermis disposed on the reactor.

[0018] FIG. 5A illustrates a synthetic tissue reactor according to the present disclosure following formation of proper dermis and epidermis on the reactor.

[0019] FIG. 5B illustrates the dermis and epidermis from FIG. 5A.

[0020] FIG. 6 provides histological hematoxylin and eosin (H&E) staining of the dermis and epidermis from FIG. 5B, showing proper formation of both dermis and epidermis.

[0021] FIG. 7 provides immunofluorescent staining with keratin 14, keratin 10 and loricrin shows specific layers of the epidermis.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0022] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0024] The singular forms “a,” “an,’‘ and “the” include plural referents unless the context clearly dictates otherwise.

[0025] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of' and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as "consisting of and "consisting essentially of the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps. along with any impurities that might result therefrom, and excludes other ingredients/steps.

[0026] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off. measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0027] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0028] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

[0029] As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” can refer to plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9-1. 1. Other meanings of “about'’ can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4.

[0030] Further, the term “comprising” should be understood as having its open- ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B can be a composition that includes A. B, and other components, but can also be a composition made of A and B only. Any documents cited herein are incorporated by reference in their entireties for any and all purposes.

[0031] Any embodiment or aspect provided herein is illustrative only and does not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more embodiments or aspects can be combined with any part or parts of any one or more other embodiments or aspects.

[0032] Each year, more than one million patients are hospitalized in the U.S. for significant skin loss due to thermal and pressure injuries, chronic diabetic ulcers or genetic skin diseases. The ability to generate engineered human skin substitutes (HSSs) has provided a promising and effective therapy for these patients (Abaci et al, 2017, Exp Biol Med). However, current commercial HSSs available from companies in the US and other companies in the world (Vig et al, 2017, Int. J. Mol. Sci.; all come as rectangular or circular planar sheets. These planar HSSs are typically grafted as multiple patches on different parts of the body, including irregular parts like fingers or facial features, requiring a high number of sutures in between the patches to cover the entire wound area.

[0033] To address this need, one can engineer 3D wearable HSS (wHSS) in custom shapes that can be directly worn on any part of the body with a regular (e.g. arms) or irregular shape (e.g. hand, face) with curved features - see U.S. patent application no. 18/115,402 and “Engineering Edgeless Human Skin With Enhanced Biomechanical Properties” Science Advances Vol. 9, Issue 4 (2023), DOI:10.1126/sciadv.ade2514, both of which are incorporated herein by reference in their entireties.

[0034] Despite its paradigm-shifting properties and clinical applications, this method required connecting the model to external pumps and tubing, the use of secondary molds, transfer of the tissue between separate containers, and complicated cell-seeding procedures, leading to a complex method of fabrication and limiting the capability to run multiple experiments simultaneously, and thus limiting the use of this technology for skin modelling and drug testing.

[0035] This invention addresses these limitations by introducing a new method that eliminates the need for external pumps or tubes, and significantly simplifies the production of stand-alone wHSS models and systems. Considering the similarities of commercial HSSs in the current market, this technology would grant a significant competitive advantage to the companies which generate autologous or allogenic skin grafts, or skin models for disease modelling or drug/cosmetics testing.

[0036] The following is illustrative, non-limiting disclosure of methods and devices according to the present disclosure, and does not limit the scope of the present disclosure or of the appended claims.

[0037] Step 1. One can first determine the shape of the skin (e.g., via 3D scanned wound area (e.g. hands)) and create 3D computer-aided drawings (CAD), which we refer to here as “Skin Scaffolds”. As the proof-of-concept, we used a rectangular shape (FIG. 1). The Skin Scaffolds can be hollow, porous and perfusable to allow for creating the desired shape and bringing the HSSs into Air-Liquid-Interface (ALI) (cell culture medium from the dermal side, and air from the epidermal side), which is required for proper formation of the epidermis. The density, size and uniform distribution of the pores on the surface of Skin Scaffold are critical to have sufficient amount of diffusion of the medium inside the scaffold to the epidermis, which is exposed to the air outside. Then, the Skin Scaffold CAD is merged with the CAD of a chamber that has two medium reservoirs that are used to recirculate the medium back and forth through the Skin Scaffold. This whole assembled system is referred to here as the “Skin Reactor” (FIG. 2A).

[0038] Step 2. We use a 3D-printer (VisiJet; Material :M2R-TN; printed by CadBlu) or use selective laser sintering (sProl40; Material: stainless-steel or titanium) to print the Skin Reactors (FIG. 2B and 2C).

[0039] Step 3. We design and 3D-print a bottom lid made of PLLA, in a rectangular shape, to temporarily close the bottom opening of the Skin Reactor (FIG. 3A).

[0040] Step 4. We coat the Skin Reactor with 8% gelatin crosslinked with 5 units/g transglutaminase at room temperature overnight. This coating prevents undesired leakage of culture medium through the pores later on when perfusion starts (FIG. 3B). [0041] Step 5. After snap-fitting the Bottom Lid to the Skin Reactor, a mixture of Collagen Type I gel and dermal fibroblasts are introduced into the Skin Reactor to fill it up. The Skin Reactor is then placed into the incubator for 1 hour to gel.

[0042] Step 6. After gelation, the Bottom Lid is removed and the Skin Reactor is submerged in fibroblast culture medium overnight for the formation and initial remodeling of the dermis. (FIG. 4). An example fibroblast culture medium can include, for example, Dulbecco's Modified Eagle Medium, Fetal Bovine Serum, Ascorbic Acid, and Penn-strep.

[0043] Step 7. The next day. 1-2 million keratinocytes in keratinocyte culture medium are seeded onto both sides of the dermis formed within the Skin Reactor (e g., around the Skin Scaffold) and incubated for 1 hour at 37 deg. C for cell attachment. The ability to seed the keratinocytes nearly immediately represents an advantage over other approaches, which approaches can require long incubation and/or waiting periods associated with keratinocyte seeding.

[0044] Step 8. After keratinocyte attachment, the assembly of Skin Reactor, dermis, and keratinocytes is submerged in skin co-culture medium for the proliferation of keratinocytes on the surface and the remodeling of the dermis for 7 days. A co-culture medium can include, for example, Dulbecco's Modified Eagle Medium/F-12, Fetal Bovine Serum, Ascorbic Acid, Epithelial growth factor, and Penn-strep.

[0045] Step 9. Then, to place the whole tissue into ALI, the medium is removed around the Skin Reactor, and 1 ml of cornification medium is filled in each reservoir. The Skin Reactor is placed onto a rocking platform (Infinity Rocker Pro, NextAdvance) at the speed of 20 cycle/min. This allows for the recirculation of the medium and perfusion of the interior of the skin, and for exposure of the outside surface to air for proper differentiation of keratinocytes and formation of the epidermis. Such medium movement can be effect without the use of a pump.

[0046] Step 10. After 10-14 days in ALL proper dermis and epidermis are formed and the wHSS is ready to use (FIG. 5). Histological H&E staining of the wHSS shows the proper formation of the dermis and epidermis (FIG. 6). Immunofluorescent staining with keratin 14, keratin 10 and loricrin shows specific layers of the epidermis (FIG. 7).

[0047] Step 11. To use wHSSs for in vitro drug testing purposes, wHSSs are kept on Skin Scaffolds under perfusion, where desired chemicals, drugs or cosmetics can be directly added to the medium or on the epidermis to mimic systemic or topical treatment, respectively.

[0048] Step 12. To explant the wHSS from the Skin Reactor, a minimal surgical incision is made longitudinally on the top surface, and wHSSs are peeled off of the Skin Scaffold. The incision site can depend on the shape of the wHSS.

[0049] Aspects

[0050] The following Aspects are illustrative only and do not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more Aspects can be combined with any part or parts of any one or more other Aspects.

[0051] Aspect 1. A synthetic tissue reactor, comprising: a reaction chamber, the reaction chamber having a porous scaffold disposed therein, the porous scaffold having an interior volume; and a first medium chamber and a second medium chamber, the synthetic tissue reactor arranged such that the interior volume of the porous scaffold is in fluid communication with the first medium chamber and the second medium chamber such that motion of the synthetic tissue reactor is configured to effect movement of a fluid among the first medium chamber, the second medium chamber, and the interior volume of the porous scaffold.

[0052] An example porous scaffold is provided in FIG. 1A. As shown, porous scaffold 100 can include a body 102, which body comprises pores 104 therethrough. Pores 104 can be of uniform size, but this is not a requirement, as the pores can comprise a range of different sizes, including a multimodal distribution of sizes. Pores can be circular in shape, but this is not a requirement, as pores can also be non-circular in shape. Body 102 can define a cross-sectional perimeter 106. Such a perimeter can be polygonal in shape, but this is not a requirement, as perimeter 106 can include curves, cut-outs, and other non-linear features.

[0053] As shown, body 102 can define an interior volume 110, and skin scaffold 102 can also include one or more channels 108. Channel 108 can place the interior volume 110 into fluid communication with the environment exterior to body 102. Channel 108 can define a polygonal cross-section - such as a square cross-section - but this is not a requirement, as channel 108 can define a cross-section that is non-polygonal.

[0054] An example such reactor is shown in FIG. 2A. As shown, synthetic tissue reactor 200 can include porous scaffold 100. Synthetic tissue reactor 200 can also include first medium chamber 202 and second medium chamber 204. Channels - such as channel 108 - can place first medium chamber 202 and second medium chamber 204 into fluid communication with the interior volume of porous scaffold 100. A medium chamber can include an aperture or other opening that engages with a channel of the porous scaffold. In this manner, motion - such as rocking motion - of the synthetic tissue reactor 200 can effect movement of a fluid among the first medium chamber 202, the second medium chamber 204, and the interior volume of the porous scaffold 100. As show n, reactor 200 can define an incubation volume 206 about porous scaffold 100. As described, one can engage a lid. bottom, or other part with the reactor such that fluid or other material introduced into the incubation volume 206 is retained there and can be released with removal or movement of the lid, bottom, or other part.

[0055] FIG. 2B provides an illustration of a synthetic tissue reactor according to the present disclosure; the reactor in FIG. 2B has been made via additive manufacturing. As shown, synthetic tissue reactor 200 can include porous scaffold 100. Synthetic tissue reactor 200 can also include first medium chamber 202 and second medium chamber 204. A channel or channels - such as channel 108 - can place first medium chamber 202 and second medium chamber 204 into fluid communication with the interior volume of porous scaffold 100. A medium chamber can include an aperture or other opening that engages with a channel of the porous scaffold. In this manner, motion - such as rocking motion - of the synthetic tissue reactor 200 can effect movement of a fluid among the first medium chamber 202, the second medium chamber 204, and the interior volume of the porous scaffold 100.

[0056] FIG. 2C provides a further example embodiment of the disclosed technology. As shown, a synthetic tissue reactor 200 can include porous scaffold 100. Synthetic tissue reactor 200 can also include first medium chamber 202 and second medium chamber 204. As shown, first medium chamber 202 and second medium chamber 204 can be present at the same end of the reactor. A channel or channels - such as channel 108 - can place first medium chamber 202 and second medium chamber 204 into fluid communication with the interior volume of porous scaffold 100. A medium chamber can include an aperture or other opening that engages with a channel of the porous scaffold. In this manner, motion - such as rocking motion, rotating motion, and the like - of the synthetic tissue reactor 200 can effect movement of a fluid among the first medium chamber 202. the second medium chamber 204, and the interior volume of the porous scaffold 100. [0057] Aspect 2. The synthetic tissue reactor of Aspect 1, wherein at least a portion of the porous scaffold conforms to a portion of a subject. The porous scaffold can, for example, be 3D printed based on a model, scan, measurement, or other representation of a subject or a portion of a subject, such as a subject’s hand or other portion of the subject in need of synthetic skin. The porous scaffold can also be insertable and/or removeable. The synthetic reactor can also be formed - for example, via 3D printing - such that the porous scaffold is part of the reaction chamber rather then being inserted later into the reaction chamber.

[0058] Aspect 3. The synthetic tissue reactor of any one of Aspects 1-2, wherein the reaction chamber defines an upper opening and a lower opening.

[0059] Aspect 4. The synthetic tissue reactor of Aspect 3, wherein the synthetic tissue reactor can further comprise a cover configured to engage with the upper opening or lower opening.

[0060] FIG. 3A depicts a bottom lid 300 useful in connection with a synthetic tissue reactors of the present disclosure. Bottom lid 300 can be engaged with a synthetic tissue reactor according to the present disclosure so as to define a fluid containment volume about the porous skin scaffold of the reactor. In this way, one can engage the bottom hd with the reactor, and the bottom lid will then act to retain fluid that is introduced to incubation volume 206 of the reactor.

[0061] FIG. 3B illustrates several synthetic tissue reactors 200 according to the present disclosure, which reactors have been coated with an example coating that includes 8% gelatin crosslinked with 5 units/g transglutaminase. As shown, bottom lid 300 has been fitted to a reactor 200, and the bottom hd thus acts to retain fluid that has been introduced to incubation volume 206.

[0062] FIG. 3C illustrates a synthetic tissue reactor of FIG. 3B engaged with a bottom lid of FIG. 3 A.

[0063] Aspect 5. The synthetic tissue reactor of any one of Aspects 1-4, further comprising at least one of an extracellular matrix material or a dermal cell type disposed on the porous scaffold. Dermal cell types can include, for example, fibroblasts, mesenchymal stromal cells, endothelial cells, or immune cells.

[0064] Aspect 6. The synthetic tissue reactor of Aspect 5, wherein the dermal cell ty pe comprises fibroblasts. [0065] Aspect 7. The synthetic tissue reactor of any one of Aspects 1-6, wherein the synthetic tissue reactor further comprises a keratinocyte superposed on the porous scaffold. The keratinocyte can be. for example, disposed on dermis. As an example, keratinocytes can be seeded onto the outer surface of dermis (for example, dermal cells and extracellular matrix). The dermis can be superposed - or even placed directly on - the porous scaffold.

[0066] Aspect 8. The synthetic tissue reactor of Aspect 6, wherein the synthetic tissue reactor further comprises an amount of epidermis superposed on the porous scaffold. The epidermis can be superposed on dermis, and the epidermis can even be disposed directly on dermis. As described elsewhere herein, the epidermis can be formed at least in part via keratinocytes.

[0067] Aspect 9. The synthetic tissue reactor of any one of Aspects 1-8, wherein the synthetic tissue reactor further comprises an amount of medium disposed in at least one of the first medium chamber and the second medium chamber. Example, non-limiting media include growth factors and cytokines.

[0068] As but one example, if a user employs endothelial cells, one can supplement the cornification medium with Vascular Endothelial Growth factor. If a user employs immune cells, one can introduce IL-6 or other cytokines. Other drugs, chemicals, and cosmetics can be included in the medium as may be appropriate for a given application or use.

[0069] Aspect 10. The synthetic tissue reactor of Aspect 9, wherein the medium comprises a cornification medium. A cornification medium can include, for example, Dulbecco's Modified Eagle Medium/F-12, Fetal Bovine Serum, CaCh, Ascorbic Acid, Epithelial growth factor, and Penn-strep.

[0070] Aspect 11. A method, comprising: forming a synthetic skin superposed on a porous scaffold disposed in a reactor, the synthetic skin comprising dermis and epidermis, the forming comprising effecting motion of the reactor so as to effect contact betw een the dermis and a medium.

[0071] Aspect 12. The method of Aspect 11, wherein the motion is effected free of pumping. Such motion can be, for example, rocking, revolution, shaking, and the like.

[0072] Aspect 13. The method of any one of Aspects 11-12, wherein the medium is a cornification medium. [0073] Aspect 14. The method of any one of Aspects 11-13, further comprising forming the dermis.

[0074] Aspect 15. The method of Aspect 14, wherein forming the dermis comprises inducing dermal cell types to remodel an extracellular matrix material. Dermal cell types can include, for example, dermal fibroblasts; dermal cells can also include mesenchymal cells, dermal papilla cells, adipocytes, sensory neurons, mesenchymal stem cells, endothelial cells, smooth muscle cells, and pericytes.

[0075] Aspect 16. The method of any one of Aspects 11-15, further comprising forming the epidermis.

[0076] Aspect 17. The method of any one of Aspects 11-16, wherein forming the epidermis comprises differentiating keratinocytes. This can be accomplished by. for example, inducing terminal differentiation of basal keratinocytes into suprabasal epidermal layers and cornified epidermal layers.

[0077] Aspect 18. The method of any one of Aspects 11-17, further comprising placing the keratinocytes into contact with ambient environment while the dermis is in contact with a medium.

[0078] Aspect 19. The method of any one of Aspects 11-18, wherein the porous scaffold conforms to at least a portion of a subject.

[0079] Aspect 20. The method of any one of Aspects 11-19, further comprising removing the skin substitute from the porous scaffold.

[0080] FIG. 4 illustrates the synthetic tissue reactor of FIG. 3C after gelation of a mixture of Collagen Type I gel and dermal fibroblasts, after the bottom lid has been removed, and after formation and initial remodeling of dermis 400 disposed on the reactor. The dermis is disposed on porous scaffold 100, which is not visible because it is covered by the dermis. Also shown are the first and second medium chamber 202 and 204. as well as channel 108 placing the first medium chamber into fluid communication with the interior volume - not shown - of porous scaffold 100.

[0081] FIG. 5A illustrates a synthetic tissue reactor according to the present disclosure, with proper dermis and epidermis 500.

[0082] FIG. 5B illustrates the dermis and epidermis 500 from FIG. 5A. [0083] FIG. 6 provides histological hematoxylin and eosin (H&E) staining of dermis 604 and epidermis 602 formed using the disclosed technology, showing proper formation of both dermis and epidermis.

[0084] Aspect 21. A method of forming a skin substitute, comprising: forming a portion of epidermis on a dermis superposed on a porous scaffold, while forming the portion of the epidermis, effecting pump-free motion of a medium in contact with the portion of dermis.

[0085] Aspect 22. The method of Aspect 21, wherein the motion communicates the medium among at least a chamber and an interior volume of the porous scaffold.

[0086] Aspect 23. The method of any one of Aspects 21-22, further comprising forming the dermis.

[0087] Aspect 24. The method of any one of Aspects 21-23, further comprising placing keratinocytes superposed on the dermis into contact with ambient environment while the dermis is in contact with the medium. As described herein, this can include exposing the keratinocytes to air while the dermis is in contact with the medium; this can in turn give rise to epidermis atop the dermis as shown herein.

[0088] Aspect 25. The method of any one of Aspects 21-24, further comprising removing the skin substitute from the porous scaffold.

Claims

What is Claimed:

1. A synthetic tissue reactor, comprising: a reaction chamber, the reaction chamber having a porous scaffold disposed therein. the porous scaffold having an interior volume; and a first medium chamber and a second medium chamber. the synthetic tissue reactor arranged such that the interior volume of the porous scaffold is in fluid communication with the first medium chamber and the second medium chamber such that motion of the synthetic tissue reactor is configured to effect movement of a fluid among the first medium chamber, the second medium chamber, and the interior volume of the porous scaffold.

2. The synthetic tissue reactor of claim 1, wherein at least a portion of the porous scaffold conforms to a portion of a subject.

3. The synthetic tissue reactor of any one of claims 1-2, wherein the reaction chamber defines an upper opening and a lower opening.

4. The synthetic tissue reactor of claim 3, further comprising a cover configured to engage with the upper opening or lower opening.

5. The synthetic tissue reactor of any one of claims 1 -2, further comprising at least one of an extracellular matrix material or a dermal cell t pe, including but not limited to fibroblasts, mesenchymal stromal cells, endothelial cells, or immune cells disposed on the porous scaffold.

6. The synthetic tissue reactor of claim 5, wherein the dermal cell type comprises fibroblasts.

7. The synthetic tissue reactor of claim 6, further comprising a keratinocyte superposed on the porous scaffold.

8. The synthetic tissue reactor of claim 6. further comprising an amount of epidermis superposed on the porous scaffold.

9. The synthetic tissue reactor of any one of claims 1-2, further comprising an amount of medium disposed in at least one of the first medium chamber and the second medium chamber.

10. The synthetic tissue reactor of claim 9, wherein the medium comprises a cornification medium.

11. A method, comprising: forming a synthetic skin superposed on a porous scaffold disposed in a reactor, the synthetic skin comprising dermis and epidermis. the forming comprising effecting motion of the reactor so as to effect contact between the dermis and a medium.

12. The method of claim 1 1, wherein the motion is effected free of pumping.

13. The method of any one of claims 11-12, wherein the medium is a cornification medium.

14. The method of any one of claims 11-13, further comprising forming the dermis.

15. The method of claim 14. wherein forming the dermis comprises inducing dermal cell types to remodel an extracellular matrix material.

16. The method of any one of claims 11-12, further comprising forming the epidermis.

17. The method of claim 1 , wherein forming the epidermis comprises differentiating keratinocytes.

18. The method of any one of claims 11-12. further comprising placing keratinocytes into contact with ambient environment while the dermis is in contact with a medium.

19. The method of any one of claims 11-12, wherein the porous scaffold conforms to at least a portion of a subject.

20. The method of any one of claims 11-12, further comprising removing the skin substitute from the porous scaffold.

21. A method of forming a skin substitute, comprising: forming a portion of epidermis on a dermis superposed on a porous scaffold, while forming the portion of the epidermis, effecting pump-free motion of a medium in contact with the portion of dermis.

22. The method of claim 21, wherein the motion communicates the medium among at least a chamber and an interior volume of the porous scaffold.

23. The method of any one of claims 21-22, further comprising forming the dermis.

24. The method of any one of claims 21-22, further comprising placing keratinocytes superposed on the dermis into contact with ambient environment while the dermis is in contact with the medium.

25. The method of any one of claims 21-22, further comprising removing the skin substitute from the porous scaffold.