Patent Application
20220211755
The disclosed subject matter relates to biomaterials derived from healthy, diseased, or transgenic region-specific tissue extracellular matrix. Particularly, the presently disclosed subject matter relates to methods to isolate, decellularize, and process regions or anatomical features of various organs from various sources (including human and animal; fetal, juvenile, and adult; healthy, diseased, and transgenic) into various formats including: acellular scaffolds, sponges, hydrogels, and solutions. The presently disclosed subject matter further relates to such scaffolds, sponges, hydrogels, and solutions suitable for stem cell culture.
Extracellular matrix (ECM) provides cells with a scaffold with tissue-specific cues (molecular, structural, biomechanical) that mediate cell function. Stem cells reside on specialized ECM niches where they remain quiescent until needed, such as stem cells in the papilla region of the kidney. Currently it is not possible to re-create the complex environment of tissues such as the kidney using synthetic materials.
Accordingly, there remains a need for a medium that provides an environment suitable for the growth of stem cells for various tissues, such as the kidney.
The purpose and advantages of the disclosed subject matter will be set forth in and apparent from the description that follows, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the methods and systems particularly pointed out in the written description and claims hereof, as well as from the appended drawings.
Native extracellular matrix (ECM) that is secreted and maintained by resident cells is of great interest for cell culture and cell delivery. As set forth below, specialized bioengineered niches for stem cells can be established using ECM-derived scaffolding materials. Although various embodiments refer to the kidney as an example, the methods and products set forth herein are applicable to various tissues. As an exemplary system, kidney is selected because of the high regional diversification of its tissue matrix. By preparing the ECM from three specialized regions of the kidney (cortex, medulla and papilla; the whole kidney, heart and bladder as controls) in three forms: (i) intact sheets of decellularized ECM, (ii) ECM hydrogels, and (iii) soluble ECM, it is shown how the structure and composition of ECM affect the function of kidney stem cells (with mesenchymal stem cells, or MSCs, serving as control). All three forms of the ECM regulate kidney stem cell (KSC) function, with differential structural and compositional effects. KSCs cultured on papilla ECM consistently display higher metabolic activity and differences in cell morphology, alignment, proliferation and structure formation as compared to cortex and medulla ECM, the effects not observed in corresponding MSC cultures. Thus, tissue- and region-specific ECM can provide an effective substrate for in vitro studies of therapeutic stem cells.
Similarly, tissues from other organs of various sources are processed into various formats. Specific regions or anatomical features of exemplary organs including the adrenal gland, bladder, blood vessel, brain, breast, bone, esophagus, heart, kidney, larynx, liver, lung, lymph node, muscle, parathyroid, pancreas, placenta, skin, small intestine, spleen, stomach, testes, thymus, thyroid, umbilical cord, and uterus are processed into various formats. These tissues are drawn from various sources including human and animal; fetal, juvenile, and adult; and healthy, diseased, and transgenic tissues. These materials are processed into formats including acellular scaffolds, sponges, hydrogels, and solutions.
To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter includes a culture medium. In some embodiments, the culture medium includes a scaffold and the scaffold comprises a decellularized extracellular matrix. In some embodiments, the scaffold comprises a substantially planar sheet. In some embodiments, the decellularized extracellular matrix is selected from the group consisting of: adrenal gland extracellular matrix, bladder extracellular matrix, blood vessel extracellular matrix, brain extracellular matrix, breast extracellular matrix, bone extracellular matrix, esophagus extracellular matrix, heart extracellular matrix, kidney extracellular matrix, larynx extracellular matrix, liver extracellular matrix, lung extracellular matrix, lymph node extracellular matrix, muscle extracellular matrix, parathyroid extracellular matrix, pancreas extracellular matrix, placenta extracellular matrix, skin extracellular matrix, small intestine extracellular matrix, spleen extracellular matrix, stomach extracellular matrix, testes extracellular matrix, thymus extracellular matrix, thyroid extracellular matrix, umbilical cord extracellular matrix, and uterus extracellular matrix. In some embodiments, the decellularized extracellular matrix is a region-specific extracellular matrix. In some embodiments, the decellularized extracellular matrix is an organ-specific extracellular matrix. In some embodiments, the decellularized extracellular matrix is extracellular matrix of a region of an organ. In some embodiments, the organ is the kidney and the region is selected from the group consisting of: cortex, medulla, and papilla.
In another aspect of the present subject matter, a kit for making a culture medium is provided. The kit includes a solution and at least one reagent. The solution comprises decellularized extracellular matrix. At least one reagent is adapted to reconstitute the solution into a hydrogel. In some embodiments, the reagent comprises phosphate buffered saline or sodium hydroxide. In some embodiments, the decellularized extracellular matrix is selected from the group consisting of: adrenal gland extracellular matrix, bladder extracellular matrix, blood vessel extracellular matrix, brain extracellular matrix, breast extracellular matrix, bone extracellular matrix, esophagus extracellular matrix, heart extracellular matrix, kidney extracellular matrix, larynx extracellular matrix, liver extracellular matrix, lung extracellular matrix, lymph node extracellular matrix, muscle extracellular matrix, parathyroid extracellular matrix, pancreas extracellular matrix, placenta extracellular matrix, skin extracellular matrix, small intestine extracellular matrix, spleen extracellular matrix, stomach extracellular matrix, testes extracellular matrix, thymus extracellular matrix, thyroid extracellular matrix, umbilical cord extracellular matrix, and uterus extracellular matrix. In some embodiments, the decellularized extracellular matrix is a region-specific extracellular matrix. In some embodiments, the decellularized extracellular matrix is an organ-specific extracellular matrix. In some embodiments, the decellularized extracellular matrix is extracellular matrix of a region of an organ. In some embodiments, the organ is the kidney and the region is selected from the group consisting of: cortex, medulla, and papilla.
In other aspects of the present subject matter, culture media are provided. In some embodiments, the culture media include a hydrogel, the hydrogel comprising decellularized extracellular matrix. In some embodiments, the culture media include solubilized decellularized extracellular matrix. In some embodiments, the culture media include a sponge, the sponge comprising decellularized extracellular matrix.
In another aspect, the disclosed subject matter includes a method of creating a hydrogel. A portion of an organ is extracted. The organ portion is decellularized to yield extracellular matrix. The extracellular matrix is powdered to yield a powder. The powder is digested to yield a digest. The digest is reconstituted into a hydrogel.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the disclosed subject matter claimed.
A detailed description of various aspects, features, and embodiments of the subject matter described herein is provided with reference to the accompanying drawings, which are briefly described below. The drawings are illustrative and are not necessarily drawn to scale, with some components and features being exaggerated for clarity. The drawings illustrate various aspects and features of the present subject matter and may illustrate one or more embodiment(s) or example(s) of the present subject matter in whole or in part.
Reference will now be made in detail to exemplary embodiments of the disclosed subject matter. Methods and corresponding steps of the disclosed subject matter will be described in conjunction with the detailed description of the system.
The extracellular matrix (ECM), the native scaffolding material secreted and maintained by residents cells, provides an ideal microenvironment for the cells with tissue-specific physical and molecular cues mediating cell proliferation, differentiation, gene expression, migration, orientation, and assembly. Functional and structural components within the ECM contribute to the extracellular environment specific to each tissue and organ. The complexity of the ECM has proven difficult to recapitulate in its entirety. Mimicking just the ECM structure using synthetic biomaterials or mimicking composition by adding purified ECM components is possible. While offering structural mimics, synthetic biomaterials can potentially generate cytotoxic by-products at the site of implantation, leading to poor wound healing or an inflammatory environment.
An alternative to synthetic biomaterials is to directly isolate the native ECM from the tissue of interest via the removal of cells and cellular remnants. ECM scaffolds may be derived from a variety of tissues such as adrenal gland, bladder, blood vessel, brain, breast, bone, esophagus, heart, kidney, larynx, liver, lung, lymph node, muscle, parathyroid, pancreas, placenta, skin, small intestine, spleen, stomach, testes, thymus, thyroid, umbilical cord, and uterus. ECM-derived biomaterials can be processed into scaffolds (such as acellular scaffolds or sponges) with appropriate compositions and structures for cell cultivation and tissue engineering. Furthermore, ECM scaffolds gradually degrade while promoting tissue remodeling at the site of implantation. Due to their biocompatibility and their ability to modulate the host tissue response, ECM scaffolds are suitable for tissue engineering and regenerative medicine applications. ECM scaffolds can be derived from various sources such as human and animal; fetal, juvenile, and adult; healthy, diseased, and transgenic tissues.
ECM-based scaffolds can also be used to regulate the differentiation and maintenance of stem cells and their differentiated progeny. Stem cells normally reside within unique and highly regulated ECM serving as a niche. Complex tissues such as heart and lung may be subjected to decellularization to obtain native-ECM scaffolds without particular regard for any specific region of the organ or preservation of potential stem cell niches. However, cells native to a particular region of the organ (e.g., vascular endothelium, liver sinusoidal cells) display ECM recognition and specificity. Extending this site-specific recognition to stem cells renders the choice of matrix an important consideration.
Referring to
The kidney is a suitable organ for studying effects of regional ECM on the resident stem cell population. A cross-sectional view of the kidney reveals three distinct regions: cortex, medulla, and papilla (
The cortex contains renal corpuscles, and the associated convoluted and straight tubules, collecting tubules and ducts, and contains an extensive vascular network. The medulla is arranged into pyramids, and characterized by straight tubules, collecting ducts, and the vasa recta, a specialized capillary system involved in the concentration of urine. At the apex of each medullary pyramid, where the collecting ducts converge and empty into the renal calyx, is the papilla. The renal papillae contain a putative population of adult stem cells that remains quiescent after the development is complete and is mobilized again during injury. This stem cell may be isolated and expanded in culture, making the kidney an excellent model to study interactions between the native stem cell population and the matrix derived from distinct regions within the organ.
ECM materials according to various embodiments of the present disclosure are useful to grow, maintain, or differentiate organ- or region-specific cells in culture. Various embodiments of the present disclosure are useful for: in vitro three-dimensional culture and testing of cells on acellular scaffolds, in sponges, or in hydrogels; in vitro culture and testing of cells with culture medium supplemented with matrix solution; in vitro coating (adsorption) of matrix solution to cell culture flask/dish to increase attachment, growth; in vitro guided differentiation of embryonic stem cells or induced pluripotent stem cells into organ-specific cells; in vivo injection/delivery of therapeutic cells, drugs, or other soluble factors via hydrogel; in vivo implantation of acellular scaffolds with or without cells for tissue/organ regeneration studies.
The present disclosure describes a method to derive regionalized ECM biomaterials, for example, for stem cell culture. Such materials include acellular scaffolds, sponges, hydrogels, and solutions. According to various embodiments of the present disclosure, materials are provided in various physical forms including various sized sheets and solubilized forms. According to various embodiments, ECM biomaterials are derived from various tissues including adrenal gland, bladder, blood vessel, brain, breast, bone, esophagus, heart, kidney, larynx, liver, lung, lymph node, muscle, parathyroid, pancreas, placenta, skin, small intestine, spleen, stomach, testes, thymus, thyroid, umbilical cord, and uterus. In some embodiments, region-specific ECM biomaterials are derived from a corresponding region in a source organ, for example, from the cortex, medulla or papilla of a kidney. Tissues sources include human and animal; fetal, juvenile, and adult; healthy, diseased, and transgenic. Described below are the regionally specific effects of kidney ECM on the growth and metabolism of kidney stem cells, how these effects depend on the preservation of ECM structure vs only composition, and extension of these effects to exogenous (non-kidney) stem cells, such as mesenchymal stem cells (MSCs).
The methods and systems presented herein may be used for creating ECM biomaterials for stem cell culture from various tissues, including region-specific kidney extracellular matrix hydrogels. The disclosed subject matter is particularly suited for creating region-specific hydrogels for the growth of stem cells, such as kidney stem cells (KSCs), and mesenchymal stem cells (MSCs).
According to embodiments of the present disclosure, native tissue matrix is used to cause region-specific effects on the growth of KSCs and mesenchymal stem cells (MSCs). To this end, hydrogels are derived from kidney regions including the cortex, medulla and papilla.
According to an exemplary method, kidneys are procured and immediately frozen and prepared for sectioning. Frozen blocks are then sectioned longitudinally into thin (200 [μm-1 mm) slices showing the entire cross-section of the kidney. The cortex, medulla, and papillae of the kidney are then dissected and separated from the thin slices prior to decellularization.
The tissues are decelluarized using a 4-step method consisting of 0.02% trypsin (2 hr), 3% Tween-20 (2 hr), 4% sodium deoxycholate (2 hr), and 0.1% peracetic acid (1 hr). Each step is followed by deionized water and 2×PBS washes. In some embodiments, each region is decellularized by serial washes in 0.02% trypsin, 3% Tween, 4% deoxycholic acid, and 0.1% peracetic acid solutions followed by enzymatic digestions.
Following decellularization, the ECMs are snap frozen in liquid nitrogen, pulverized using a mortar and pestle, and then lyophilized to obtain a fine powder. Lyophilized ECM powder is digested using pepsin and hydrochloric acid for 48 hours at room temperature. The resulting digest is re-constituted into a hydrogel by increasing the ionic strength and the pH of the solution using PBS and NaOH.
The results are highly specific hydrogels composed of the native extracellular matrix surrounding native cells. They may be used to grow and maintain tissue-specific cells in culture. In some embodiments, cells are cultured on the hydrogels. In other embodiments, cells are cultured in media supplemented with digested ECM. Metabolic activity, image analysis and DNA quantification may be performed.
In one embodiment, kidney stem cells isolated from the papilla are maintained by culturing the cells in papilla derived ECM hydrogels in vitro. Hydrogels may also be used as an injectable therapeutic platform for the delivery of drugs and/or cell therapy to an injured kidney or to guide the differentiation of embryonic stem cells or induced pluripotent stem cells into kidney specific cells for renal tissue engineering applications.
KSCs cultured in the presence of papilla ECM show higher metabolic activity and lower DNA content when compared to whole kidney, cortex and medulla ECM, an effect not observed using MSCs. Thus, the hydrogels derived from the native kidney ECM stimulate the parent KSCs but not the MSCs. Region specific kidney ECM affects the growth and metabolism of KSCs. Region-specific ECM thus provides a suitable substrate for cultivation and delivery of stem cells and their derivatives.
According to various embodiments of the present disclosure, ECM is extracted from organs and tissues including the adrenal gland, bladder, blood vessel, brain, breast, bone, esophagus, heart, kidney, larynx, liver, lung, lymph node, muscle, parathyroid, pancreas, placenta, skin, small intestine, spleen, stomach, testes, thymus, thyroid, umbilical cord, and uterus. Organs/tissues are procured, prepared for sectioning, frozen, then sectioned into thin slices. In some embodiments, the slices are about 200 μm to about 1 mm thick. Organ regions, sub-sections, or anatomical features of interest are further dissected and separated prior to decellularization. In various exemplary embodiments, region-specific tissues are extracted from the kidney cortex, medulla, or papilla; the lung airways or parenchyma; the esophageal endomucosa or muscularis externa; or the heart ventricle or atrium.
Tissue sections are decelluarized by the introduction of one or more of deionized water, hypertonic salines, enzymes, detergents, and acids. In an exemplary embodiment, heart ventricle sections are decellularized by 0.02% trypsin (2 hr), 3% Tween-20 (2 hr), 4% sodium deoxycholate (2 hr), and 0.1% peracetic acid (1 hr). Each step is followed by deionized water and hypertonic (2×) phosphate-buffered saline (PBS) washes. Exemplary embodiments for various organs and tissues of human and animal origin are provided below in Table 1.
Following decellularization, resulting materials are terminally sterilized and biopsied according to desired scaffold size. In some embodiments, the scaffold is sized to fit in the wells of a standard a microtiter plate, for example a 24- or 96-well plate.
In some embodiments, following decellularization, an ECM solution is produced. The decellularized material is snap frozen in liquid nitrogen, pulverized using a mortar and pestle, lyophilized, and then milled to obtain a fine ECM powder. In some embodiments, the ECM powder is digested using 1 mg/mL pepsin and 0.1M hydrochloric acid for more than 24 hrs at room temperature. The resulting digest is neutralized, frozen, and thawed to obtain ECM solution.
In some embodiments, ECM powder is further processed to form an ECM sponge. ECM powder is digested using 1 mg/mL pepsin and 0.1M hydrochloric acid for less than 24 hrs at room temperature. The resulting digest is subjected to repeated cycles of high-speed centrifugation (5,000 rpm) and vortexing. The resulting material is transferred to a mold of desired dimensions and lyophilized. The resulting sponge can be sectioned, re-sized, or re-hydrated. In some embodiments, the sponge is sized to fit in the wells of a standard a microtiter plate, for example a 24- or 96-well plate.
In some embodiments, ECM solution is ECM solution is re-constituted into a hydrogel by increasing the ionic strength and the pH of the solution using PBS and NaOH.
Composition and Gelation Properties of Decellularized Kidney ECM
Referring to
Decellularization of kidney regions (cortex, medulla, papilla) by a four step method (trypsin, Tween 20, sodium deoxycholate, peracetic acid) resulted in the removal of >99% nuclear material as shown by H&E staining and DNA quantification (
Electrophoresed kidney region ECM digests and purified collagen I showed major bands at similar locations, indicating that collagen I is a large component of the kidney region ECM digests, with other bands distinct from pure collagen I (
Ultrastructure of Native and Decellularized Kidney ECM
Referring to
Native and decellularized kidney regions were imaged via SEM to investigate preservation of the ultrastructure after decellularization (
Collagen IV and Fibronectin in Native and Decellularized Kidney ECM
Referring to
Native and decellularized kidney regions were immunostained to reveal the amounts and distributions of collagen IV and fibronectin in kidney regions before and after decellularization (
DNA and Metabolic Activity of KSCs in Solubilized Kidney ECM
Referring to
KSCs and MSCs were cultured on tissue culture plastic in media supplemented with solubilized ECM derived from the three kidney regions or the whole kidney. DNA and metabolic activity were measured and expressed relatively to the corresponding values measured for cells grown in media supplemented with purified solubilized collagen I (
No significant differences in metabolism were observed between KSCs in solubilized cortex and medulla ECM (
DNA, Metabolic Activity, and Phenotype of KSCs on Regional Kidney ECM Hydrogels
Referring to
KSCs and MSCs were seeded at equal densities on decellularized kidney ECM hydrogels (
Metabolic activity per unit DNA again indicated that KSCs on papilla ECM hydrogel were significantly more metabolically active than were KSCs on whole kidney, cortex or medulla ECM hydrogel. No significant differences were observed in metabolism between KSCs on cortex and medulla hydrogels (
DNA, Metabolic Activity, and Phenotype of KSCs on Regional Kidney ECM Sheets
Referring to
KSCs and MSCs were seeded at equal densities on decellularized kidney ECM sheets (
DNA quantification of KSCs cultured on decellularized ECM sheets revealed significant differences between cortex, medulla, and papilla regions, with papilla ECM again yielding the fewest KSCs (
In addition to differences in cell number, distinct morphologies and orientation of KSCs were observed in cortex, medulla, and papilla ECM (
Structure Formation by KSCs on Kidney ECM Sheets
Referring to
KSCs were seeded at equal densities onto ECM sheets derived from decellularized kidney regions (cortex, medulla, and papilla), cultured for 48 hrs or 7 days, and imaged. KSCs showed clear differences when cultured on ECM sheets from different kidney regions in cell morphology, orientation, and structure formation already by 48 hrs of cultivation (
Metabolic Activity of KSCs on Whole Organ ECM
Kidney stem cells (KSCs) were seeded onto tissue culture plastic and cultured for 48 hrs in three different forms of ECM (decellularized sheets, hydrogels, and solubilized forms) obtained from porcine hearts, bladders and kidneys. KSCs grown on decellularized whole kidney sheets and whole kidney hydrogel showed significantly higher metabolic activity at 48 hrs when compared to KSCs grown on bladder and heart ECM sheets and hydrogels (
Chemotaxis (Transwell) Assay
KSCs seeded onto transwells with 8 μm pores were cultured in the presence of solubilized kidney region ECM (
The present disclosure provides ECM biomaterials in various formats including acellular scaffolds, sponges, hydrogels, and solutions. These materials are derived from various tissues such as adrenal gland, bladder, blood vessel, brain, breast, bone, esophagus, heart, kidney, larynx, liver, lung, lymph node, muscle, parathyroid, pancreas, placenta, skin, small intestine, spleen, stomach, testes, thymus, thyroid, umbilical cord, and uterus. Tissues may be from various sources such as human and animal; fetal, juvenile, and adult; healthy, diseased, and transgenic. These ECM biomaterials modulate stem cells in a region-specific manner. For example, data show that there is a significant degree of recognition and specificity between adult kidney stem cells and their extracellular environment. KSCs showed significantly higher proliferation and higher metabolic activity in kidney ECM when compared to KSCs in ECM from other organs (
Kidney stem cells cultured on whole kidney ECM were compared to ECM derived from the urinary bladder and heart to determine if there was recognition between KSCs and the ECM at the organ level. ECM from whole bladder, heart, and kidney was prepared in three different forms: decellularized sheets, hydrogels, and solubilized forms. KSCs were significantly more proliferative and metabolically active in all three forms of kidney ECM when compared to respective forms of bladder or heart ECM (
The organ specificity of KSCs according to the present disclosure demonstrates specificity of liver sinusoidal endothelial cells to liver ECM and indicates that decellularized kidney ECM sheets contain organ-specific cues. Higher KSC metabolism is observed in whole kidney ECM hydrogel and soluble ECM, where the ECM ultra-structure is absent and only a homogenous mix of digested ECM proteins (cross-linked in the hydrogel or dissolved in solution) comprises the extracellular environment (
A degree of kidney stem cell-matrix specificity has been shown at the organ level. Accordingly methods are provided to isolate and prepare ECM biomaterials from three distinct regions of the kidney—the cortex, medulla, and papilla—to show cell-matrix interactions at the regional level. Each region of the kidney harbors a variety of cell types and structures, including extensive networks of tubules, collecting ducts, and capillaries, necessary for filtering blood or concentrating urine. In an adult mouse kidney, label-retaining cells (KSCs) remain quiescent in the renal papilla (stem cell niche) and migrate to the site of injury following renal ischemia. Consequently, this adult kidney stem cell population may be used to investigate region-specific effects of kidney ECM on the proliferation and metabolism of KSCs.
Characterization of the ECM in native cortex, medulla, and papilla reveals significant differences in structure and composition, many of which are retained after decellularization and further processing. Following the removal of >99% nuclear material (
Scanning electron micrographs of kidney region ECM showed comparable topographies between native and decellularized sections, indicating that many ultra-structural features of the ECM are retained after cells are removed. Additionally, large tubular collecting ducts approximately 50 [tm in diameter are seen in decellularized papilla sections (
While structural cues account for some organ or even region-specific signaling to kidney stem cells, compositional cues from the ECM also play a role in informing KSCs about their extracellular environment. Decellularized whole mouse kidney ECM are able to direct the differentiation of embryonic stem cells into specialized cells types as well as to encourage proliferation along the basement membrane, indicating that the basement membrane or one or more of its components promotes signaling for proliferation. Differences in the composition and distribution of the basement membrane in different regions of the kidney thus account for some of the region-specific differences observed in KSC proliferation and metabolism. Further, kidney region ECM biomaterials may be used to selectively differentiate KSCs into region-specific cell types.
As shown in
Across the ECM regions discussed above, KSCs cultured in papilla ECM consistently showed significantly lower cell number (DNA content) when compared to KSCs in cortex and medulla ECM (
When cultured in ECM obtained from entire kidney sections (containing cortex, medulla, and papilla), the metabolic activity was found to be within the values obtained for the individual regions, suggesting a dose effect. In addition, factors may be more readily available in the solubilized form but may still be locked into place or obscured by other proteins in an intact decellularized sheet.
One aspect of this work is the development of tissue-specific biomaterials and the potential for tissue regeneration using regionalized ECM biomaterials to direct the differentiation of reparative stem cells, for example to address renal pathologies such as diabetes or kidney failure. This approach translates into other regionalized organs as well. Cultivation of epithelial and endothelial cells on fully decellularized rat kidney scaffolds in a whole-organ perfused bioreactor results in a bioengineered kidney that produced rudimentary urine in vitro (in the bioreactor) and in vivo (following orthotopic implantation in rat). A variety of cell and tissue engineering applications may be applied in conjunction with regionalized ECMs. For example, since the renal papilla is the KSC niche, it may be used to maintain the cells in a stem-like state in vitro, while cortex and medulla ECM may be used to differentiate KSCs into other renal cell types.
Ischemic conditions in the cortex encourage mobilization, migration, and differentiation of quiescent KSCs in the papilla. The data in
With regard to
With regard to
With regard to
ECM biomaterials derived from the tissues affect the growth and metabolism of stem cells with regional specificity. Region-specific ECM may thus provide an optimal substrate for the in vitro cultivation or the delivery of therapeutic stem cells and their derivatives. The present disclosure is application to development of biomaterials or applications in tissue repair and regeneration using regionalized ECM biomaterials to deliver and direct the differentiation of reparative stem cells to address pathologies such as diabetes or kidney failure.
Decellularization
Porcine bladders, hearts, and kidneys were procured from Yorkshire pigs (65-70 kg) immediately following euthanasia, excess tissue was trimmed, and the blood and debris removed with water. The organs were stored at −80° C. for at least 24 hrs, thawed and then sliced into <2 mm thin cross-sections. Cross-sections from the middle third of the kidney were separated into cortical, medullary, and papillary regions. Whole kidneys and kidney regions were decellularized using a modification of a previously established method. Briefly, the slices were washed with 2× phosphate-buffered saline (PBS) for 15 min, followed by 2 hrs of 0.02% trypsin, 2 hrs of 3% Tween-20, and 2 hrs of 4% sodium deoxycholate treatment. After each step, kidney sections were washed with 2×PBS for 15 min. Kidney ECM slices were treated for 1 hr with 0.1% peracetic acid and subjected to alternating sterile 1×PBS and dH2O washes.
Histological Analysis
Native and decellularized tissue samples were fixed in formalin, embedded in paraffin, sectioned at 5 ium thickness, stained with hematoxylin and eosin, Trichrome, or Alcian Blue, and imaged using an Olympus IX81 microscope at 10×.
DNA Quantification of Decellularized Tissue
DNA content of decellularized tissue was quantified using Quanti-iT PicoGreen dsDNA Assay kit (Invitrogen) according to the manufacturer's instructions. Briefly, tissue samples were weighed, digested overnight with Proteinase K in TEX buffer at 56° C., and mixed with PicoGreen reagent. Fluorescence emission was measured at 520 nm with excitation at 480 nm, and DNA was quantified using a standard curve.
ECM Characterization
Collagen content of kidney region sheets/digests was determined using the Sircol collagen assay kit (Biocolor). Samples were digested in 0.1 mg pepsin/mL overnight at room temperature (25° C.), and the Sircol assay was performed according to the manufacturer's instructions. Sulfated glycosaminoglycan (sGAG) content of kidney region sheets/digests was determined using the 1, 9-dimethylene blue (DMB) dye binding assay. Samples were digested in 125[Lg papain/mL overnight at 60° C. sGAG content was quantified by mixing ECM digest samples with DMB dye in a 1:5 ratio and reading spectrophotometric absorbance at 595 nm and 540 nm. The difference in absorbance at these wavelengths was used with a chondroitin-6-sulphate standard curve to quantify sGAG content. Pepsin digests of the regional kidney ECM and collagen I (BD, Biosciences) were electrophoresed on 7.5% polyacrylamide gels (BioRad) under reducing conditions (5% 2-mercaptoethanol). The proteins were visualized with Coomassie Brilliant Blue (BioRad) and imaged by scanning the polyacrylamide gel.
Scanning Electron Microscopy (SEM)
Native and decellularized sections of regional kidney ECM were fixed in formalin, rinsed in 70% EtOH, frozen, lyophilized, and gold-coated (5 nm thickness). Sections were imaged on a Hitachi S-4700 FE-SEM with accelerating voltage 2.5 kV.
Immunohistochemical Staining
Sections of native and decellularized kidney ECM were fixed in formalin for 30 min, embedded in paraffin, cut to 5 μm, and mounted on slides. Sections were deparaffinized and subjected to boiling citrate buffer (pH=6.0) for 16 minutes for antigen retrieval, and blocked with 10% Normal Goat Serum in PBS for 2 hrs at room temperature. Primary antibody staining was performed for 2 hrs at 4° C. using the following primary antibodies and dilutions: Collagen IV (Rb pAb to Coll IV, ab6586) diluted 1:200 and Fibronectin (Rb pAb to Fibronectin (ab23750)) diluted 1:200. For all stains, the secondary antibody (Goat pAb to Rb IgG (ab98464)) was diluted 1:200 and incubated for 1 hr at room temperature. Sections were mounted in Vectashield Mounting Medium with DAPI, cover slipped, and imaged with an Olympus IX81 microscope at 10×.
Mouse Kidney Stem Cells
Mouse kidney stem cells (KSCs) were obtained from mouse kidneys as previously described, cultured in Dulbecco's Modified Eagle Medium (DMEM) with high glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under standard culture conditions (37° C. and 5% CO2).
Mouse Mesenchymal Stem Cells
Mouse mesenchymal stem cells (MSCs) were obtained from Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine and cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS, 10% horse serum, and 1% penicillin/streptomycin under standard culture conditions (37° C. and 5% CO2).
Preparation of ECM Sheets, ECM Hydrogels, and Solubilized ECM
Decellularized whole organ and regional kidney slices were either perforated with a 7 mm biopsy punch into sheets or snap-frozen in liquid nitrogen. Sheets were stored in 1×PBS at 4° C. until use while frozen pieces were pulverized into a fine powder using a mortar and pestle, and lyophilized for 24 hrs. Lyophilized ECM powder was digested as previously described. Briefly, 1 g of lyophilized ECM powder was mixed with 0.1 g pepsin (Sigma, ˜2500 U/mg) in 0.01M HCl. The solution was allowed to digest for 48 hrs at room temperature (25° C.) under constant stirring. Final digests were aliquotted and stored at −80° C. until use. The soluble ECM was obtained by neutralizing ECM stock digests and added to cell culture media directly (Typically 1 mg dry ECM/ml medium). Hydrogels were prepared as previously described by mixing ECM stock digests with 1×PBS, 10×PBS, and 0.1M NaOH to yield a hydrogel with a final concentration of 6 mg/mL at 4° C.
Solubilized Mitogenicity Assay
KSCs and MSCs were seeded on tissue culture plastic (TCP) at 2.5×104 cells/mL, cultured for 24 hrs in media supplemented with 10% FBS, and starved for 24 hrs in media containing 0.5% FBS. Next, ECM digests were added to the media (0.1 mg/mL) with 0.5% FBS for 48 hrs. On the fourth day, culture media was replaced with media containing 10% Alamar Blue® (Invitrogen) and the cells were incubated for 14 hrs. Culture media were transferred into new 96-well plates and absorbance was measured at 570 nm and normalized to 600 nm.
DNA Quantification of Seeded ECM Sheets and Hydrogels
DNA content of seeded ECM sheets and hydrogels was quantified using Quanti-iT PicoGreen dsDNA Assay kit (Invitrogen) according to the manufacturer's instructions. After 48 hrs of culture, samples were digested in 125 μg papain/mL overnight at 60° C. and mixed with PicoGreen reagent. Fluorescence emission was measured at 520 nm with excitation at 480 nm, and DNA was quantified using a standard curve.
Metabolic Activity
ECM sheets were glued to the bottom of 96-well plates using 2% fibrin. ECM and collagen I hydrogels were prepared in 96-well plates by adding 501 μL of hydrogel (neutralized and brought to the concentration of 6 mg/ml) at 4° C. The plates containing the hydrogels were incubated for 40 minutes at 37° C. until gelation was observed. KSCs and MSCs were grown under standard culture conditions, trypsinized, seeded into the ECM sheets or hydrogels at 2.5×104 cells/mL, and cultured for 48 hr or 7 days. After a 48-hr incubation, the culture media was replaced with media containing 10% Alamar Blue® (Invitrogen). After 14-hr incubation, media were transferred into new 96-well plates and absorbance was measured at 570 nm and normalized to 600 nm.
Confocal Imaging
KSCs and MSCs grown under standard culture conditions were seeded into ECM sheets or hydrogels at 2.5×104 cells/mL and cultured for 48 hrs or 7 days, at which times they were stained with Live/Dead Viability Kit (Invitrogen) or fixed with 3.7% formaldehyde and stained with rhodamine phalloidin (Invitrogen) and DAPI according to the manufacturer's instructions. Confocal imaging was performed using an Olympus Fluoview FV1000 Confocal Microscope.
Gelation Kinetics
Regional kidney ECM hydrogels and collagen I hydrogels were prepared as previously described. Gelation kinetics were determined spectrophotometrically as previously described. Briefly, gel solutions at 4° C. were transferred to a cold 96-well plate by adding 100 μL/well in triplicate. The SpectraMax spectrophotometer was pre-heated to 37° C., plate was loaded, and turbidity measured at 405 nm every 2 min for 1.5 hrs. Absorbance values were recorded for each well and averaged. Three separate tests were performed on two separate batches of kidney ECM hydrogels.
Chemotaxis (Transwell) Assay
KSCs were cultured for 24 hrs in 0.5% FBS, trypsinized, and seeded onto transwells with 8 ium pores. Region solubilized kidney ECM was added to the media at a concentration of 0.1 mg/mL. After 6 hrs, transwells were collected, attached cells removed from the top of the membrane using a Q-tip, and membranes were detached. DNA from cells attached to the bottoms of the detached membranes was quantified with CyQuant® Direct Cell Proliferation Assay Kit according to the manufacturer's instructions. Fluorescence emission was measured at 535 nm with excitation at 480 nm, and DNA was quantified using a standard curve.
Statistical Analysis
One-way ANOVA test with Tukey's multiple comparison post hoc test and two-way ANOVA test with Bonferroni post hoc test were performed using Prism v6 (GraphPad, La Jolla Calif.). A p<0.05 was considered statistically significant.
While the disclosed subject matter is described herein in terms of certain exemplary embodiments, those skilled in the art will recognize that various modifications and improvements may be made to the disclosed subject matter without departing from the scope thereof Moreover, although individual features of one embodiment of the disclosed subject matter may be discussed herein or shown in the drawings of the one embodiment and not in other embodiments, it should be apparent that individual features of one embodiment may be combined with one or more features of another embodiment or features from a plurality of embodiments.
In addition to the specific embodiments claimed below, the disclosed subject matter is also directed to other embodiments having any other possible combination of the dependent features claimed below and those disclosed above. As such, the particular features presented in the dependent claims and disclosed above can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter should be recognized as also specifically directed to other embodiments having any other possible combinations. Thus, the foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.
It will be apparent to those skilled in the art that various modifications and variations can be made in the method and system of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter include modifications and variations that are within the scope of the appended claims and their equivalents.
This application is a continuation application of U.S. application Ser. No. 15/913,237, filed Mar. 6, 2018, which is a continuation application of U.S. application Ser. No. 14/450,020, filed Aug. 1, 2014, which claims the benefit of U.S. Provisional Application No. 61/861,958, filed Aug. 2, 2013 and claims the benefit of U.S. Provisional Application No. 61/862,933, filed Aug. 6, 2013.
This invention was made with government support under grant number EB002520 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61862933 | Aug 2013 | US | |
| 61861958 | Aug 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15913237 | Mar 2018 | US |
| Child | 17696321 | US | |
| Parent | 14450020 | Aug 2014 | US |
| Child | 15913237 | US |
