The difference between these two isoforms is that ECM1b lacks amino acids You however wouldn't be able to specifically detect ECM1b as all the amino acids present in this isoform are also present in ECM1a. Do you synthesize the anti-sera based on DNA sequence of proteins, if you do not, can you suggest me a company they make anti-sera, many thanks! Thank you for contacting us. In general, the selection of the immunogen varies from antibody to antibody. Some antibodies are raised against synthetic peptides based ona protein sequence which was derived originally from a DNA sequence , some against protein fragments and others against the full length protein either purified or overexpressed in another organism or a cell preparation.
I agree to the terms and conditions. Immunology Immunoglobulins Receptors. Datasheet References 2 Protocols Overview. IP: A cells. FC: A cells. Concentration information loading Associated products. Recombinant Human Extracellular matrix protein 1 ab Involved in endochondral bone formation as negative regulator of bone mineralization. In some instances, the composition of the present i nvention is configured as a delivery vehicle for therapeutic agents, cells, protei ns, or other biological materials.
In one embodiment, the composition of the present invention can be used to deliver platelet-rich plasma PRP that is derived from whole blood of the patient or from another blood donor. The cells that can be delivered by the composition of the present invention include, but are not limited to, plunpotent or multipotent stem cells, mesoderm precursor cells, adipocytes, lipoblasts, or precursors thereof, e.
In some instances, the composition is configured to coat surfaces, such as tissue culture plates or scaffolds, to culture adipocytes and lipoblasts or other cell types, such as adipose-derived mesenchymal stem cells, or other adipocyte progenitors relevant to adipose tissue repai r and research. The composition of the present invention can encourage adipogenesis of stem cells injected with it, as wel l as stem cells natural ly present in the injection region. In some instances, the decel lularized and delipidized adipose matrix of the present invention can also be used to coat implanted devices or materials to improve adipogenesis or biocompatibi!
The inventive method comprises the following steps: obtaining an adipose tissue sample e. The invention further comprises treating the decellularized adipose or loose connective tissue extracellular matrix with one or more delipidizing agents, such as lipase and colipase, or other enzymes, to obtain decellularized and delipidized extracellular matrix. Finally, the method can include sterilizing the resulting 5 decellularized and delipidized extracellular matrix.
In some instances, the methods and use of detergents and lipase can also be utilized to decellularize and delipidize other tissue components that have lipids, such as skeletal muscle, heart, or liver. In some instances, the method further comprises the step of enzymatically treating e. In some instances, the method further comprises the step of re-lyophilizing the extracellular matrix solution and then rehydrating prior to injection or implantation.
In some instances, the solution is a phosphate buffered solution PBS or saline solution which can be injected through a 25 gauge needle or smaller into the adipose tissue. In some instances, said composition further comprises cells, drugs, proteins or other therapeutic agents that can be delivered within or5 attached to the composition before, during or after gelation.
In some instances, the present invention also provides a method of encouraging adipogenesis of stem or progenitor cells injected or naturally present in the injection region using the decellularized and delipidized adipose or loose connective tissue extracellular matrix. In some instances, the present invention also provides a method of improving biocompatibility around implanted devices by coating the implanted devices with the decellularized and delipidized adipose or loose connective tissue extracellular matrix.
In some instances, the exogenous cells are adipocytes, lipoblasts, adipose-derived mesenchymal stem cells, adipose cell progenitors, and any other cell types relevant to adipose tissue repair or regeneration. Human lipoaspirate was processed to remove both cellular and lipid content. Raw lipoaspirate Figs. Removal of lipids using lipase produced a white ECM, free of cellular and lipid content Figs.
Oil red O staining Figs. I G, 1 H, 1 1 confirmed the removal of lipids. A DNEasy assay quantified the remaining nuclear content after decellularization and delipidization of the lipoaspirate. Decellularized and delipidized adipose matrix produced a dry, white powder Fig. This solubilized adipose matrix was induced to self-assemble Fig. As compared to a collagen control lane C , gel electrophoresis revealed collagen as well as multiple lower molecular weight peptides present within adipose matrix that had been decellularized using SDS lane A or sodium deoxycholatc lane B.
Protein ladder lane D was run with peptide weights in kDa. Fluorescent antibody staining of both fresh human lipoaspirate Fig. Laminin was also present in both cases, but there was some loss of content as a result of the decellularization. SEM images of adipose matrix gels revealed a porous structure composed of intermeshed fibers with a diameter of approximately nm. Live Dead analysis after 14 days in culture revealed negligible cell death of hASCs seeded on normal tissue culture plastic Fig.
Cells growing on the adipose matrix also exhibited a healthy fibroblast- like phenotype Fig. PicoGreen analysis at various time points indicates that the adipose ECM promoted normal proliferation over 2 weeks in culture Fig. Each group increased significantly between time points but no significant difference was found between groups at each time point. Solubilized adipose matrix was injected subcutaneous! The solubilized ECM formed a solid bolus beneath the skin within 1 5 minutes Fig.
Gels held their shape when excised Fig.
Bioprinting of 3D Tissue Models Using Decellularized Extracellular Matrix Bioink
The composition of the present invention can be used, for example, to support regeneration of adipocytes and to deliver therapeutic agents, including exogenous cells, into the tissue of a subject in need of therapeutic tissue engineering, filling soft tissue defects, or cosmetic and reconstructive procedures. The extracellular matrix of the invention can also be adapted for culturing cells ex vivo for further research or commercial purposes.
The extracellular matrix of the present invention can be derived from the native or natural matrix of adipose, loose connective tissue or odier tissues that contain adipocytes. The decellularized and delipidized extracellular matrix retains at least some native peptides and glycosaminoglycans which support regeneration of adipocytes. The decellularized and delipidized extracellular matrix retains at least some native peptides and glycosaminoglycans which support biological activity, such as regeneration of adipocytes or other bodily repair response.
The adipose or loose connective tissue extracellular matrix of the present invention can also be used to recruit the patients' cells into the injured tissue or as a cell or drug delivery vehicle, and can also be used to support injured tissue or change the mechanical properties of the tissue. Adipose or loose connective tissue extracellular matrix as described herein is derived from adipose or loose connective tissue, or other tissues containing adipocytes and lipids.
The adipose or loose connective tissue extracellular matrix material can be used for autologous, allogenic or xenogenic treatments. By using decellularized and delipidized extracellular matrix, the composition mimics the extracellular environment present in adipose tissue such as by providing certain proteins such as collagens 1, III and IV and glycosaminoglycans such as laminin. The invention encourages the migration of host progenitor cells that will regenerate new adipose tissue in vivo and aid integration with the existing tissue.
The composition can also be modified to encourage biological processes such as angiogenesis by attaching growth factors to the binding receptors inherently present in the remaining extracellular matrix, which will enhance this new tissue formation. An extracellular matrix composition herein can further comprise one or more additional components, for example without limitation: platelet-rich plasma PRP derived from whole blood, an exogenous cell, a polypeptide, a protein, a vector expressing a DNA of a bioactive molecule, and other therapeutic agents such as drugs, cellular growth factors, chemotaxis agents, nutrients, antibiotics or other bioactive molecules.
Therefore, in certain preferred embodiments, the extracellular matrix composition can further comprise an exogenous population of cells such as adipocytes, lipoblasts, or precursors thereof, as described below. In some instances, the composition comprising the adipose extracellular matrix herein can recaiit endogenous cells within the recipient and can coordinate the function of the newly recruited or added cells, allowing for cell proliferation or migration within the composition.
In particular, the invention relates to a biocompatible composition comprising decellularized and delipidized extracellular matrix derived directly from lipoaspirate obtained from surgical liposuction of an adipose tissue. The composition can be used for treating defective, diseased, or 1 I damaged adipose tissue, loose connective tissues, or soft tissues or organs in a subject, including a human, by injecting or implanting the biocompatible composition compri sing the decellularized and delipidized adipose extracellular matrix into the subject.
Other embodiments of the invention concern decellularized and delipidized loose connective tissues containing adipocytes and lipids, extracellular matrix compositions made therefrom, methods of use and methods of production. In some embodiments, the biocompatible composition comprising the decellularized and delipidized adipose or loose connective tissue extracellular matrix is prepared into an injectable solution form, and can be used for adipose tissue or connective tissue repair by transplanting or delivering therapeutic agents or cells contained therein into the defecti ve, diseased, or damaged tissues, or recruiting the patient's own cell s into the extracellular matrix of the invention.
In other instances, the biocompatible material comprising a decellularized and delipidized adipose or loose connective tissue extracellular matrix is, for example incorporated into another bodily implant, a patch, an emulsion, a viscous liquid, particles, microbeads, or nanobeads. Methods for manufacturing and coating a culture surface, such as tissue culture plates or wells, with decellularized and deli pidized adipose or loose connective tissue extracellular matrix are also provided. The biocompatible materials of the invention are also suitable for implantation into a patient, whether human or animal.
An appropriate digestion and preparation protocol is provided that can create nanofibrous gels. The gel solution is capable of being injected or surgically implanted into the adipose or loose connective tissue, thus demonstrating its potential as an in situ gelling scaffold. The decellularized, delipidized, and solubilized extracellular matrix of the present invention can also be gelled ex vivo, modified and shaped if desired, and then i mplanted as a three-dimensional scaffold. Since a decellularized and delipidized adipose tissue extracellular matrix mimics the natural adipose or loose connective tissue environment, it improves cell survival and retention at the site, thus encouraging adipose or loose connective tissue regeneration.
The resulting decellularized and delipidized extracellular matrix can be used as a material for adipose tissue engineering, filling soft tissue defects, and cosmetic and reconstructive surgery as non-limiting examples. The composition can be injected in particulate form or digested to create a solution that reassembles into a gel after injection.
Extracellular Matrix Protocols by Charles Streuli, Michael Grant - hitovaru.tk
Implantation of the intact matrix as a gel formed, modified, and shaped ex vivo, is also possible. The decellularized and delipidized adipose extracellular matrix can also be used as a substrate to culture adipose derived stem cells, as well as other stem or progenitor cells, for research and commercial expansion. The method comprises the following steps: obtaining an adipose tissue sample having an extracellular matrix component and non-extracellular matrix adipocyte component; treating the adipose tissue sample with one or more decellularization detergent agents, such as sodium dodecyl sulfate SDS and sodium deo ycholate, to obtain decel lularized adipose or loose connective tissue extracellular matrix, including extracellular proteins e.
An alternation of hypertonic and hypotonic solutions could also be used, alone or in combination, with the above agents for decellularization. The compositions comprise an adipose tissue extracellular matrix that is decellularized in that the majority of living cells l in the adipose or loose connective ti ssue are removed.
The amount of decellularization can be determined indirectly through an analysis of DNA content remaining in the decellularized adipose extracellular matrix, as described herein. The compositions comprise a decellularized matrix that is also substantially delipidized in that the majority of the lipids in the adipose or loose connective tissue are removed. The amount of delipidization can be determined indirectly through an oil imagine staining or a visual inspection of the whitening of the tissue, as described herein.
Development of human extracellular matrix for regenerative medicine
The ground extracellular matrix can be solubilized with an aqueous solution such as water or saline, for example. In some embodiments, the0 extracellular matrix can be solubilized at a low pH, between about pH 1 -6, or pH 1 -4 such as through addition of HO. In some embodiments, the matrix is digested with pepsin or alternative matrix peptide or glycosaminoglycan digesting enzymes, such as papain, matrix metalloproteinases, collagenases, and trypsin. In some instances, the method further comprises the step of re-lyophilizing the extracellular matrix solution, and then rehydrating in an aqueous solution prior to injection or implantation.
The solution comprising the adipose or loose connective tissue extracellular matrix can then be injected through a needle, such as 25 gauge or smaller, into the desired site of a subject in need. Decellularized and delipidized extracellular matrices are prepared such that natural or enhanced bioactivity for the adipose or loose connective tissue matrix is established.
In some instances, a polymer added to the composition is biocompatible, biodegradable or bioabsorbable. Exemplary biodegradable or bioabsorbable polymers include, but are not limited to: polylactides, poly-glycolides, polycarprolactone, polydioxane and their random and block copolymers. A biodegradable or bioabsorbable polymer can contain a monomer selected from the group consisting of a glycolide, lactide, dioxanone, caprolactone, trimethylene carbonate, ethylene glycol and lysine.
Other examples of suitable biocompatible polymers are polyhydroxyalkyl methacrylates including ethylmeth aery late, and hydrogels such as polyvinylpyrrolidone and polyacrylamides. Other suitable bioabsorbable materials are biopolymers which include collagen, gelatin, alginic acid, chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acids, polylysine and copolymers of these materials.
Any combination, copolymer, polymer or blend thereof of the above examples is contemplated for use according to the present invention. In 5 another non-limiting embodiment the gel can be injected at body temperature, but gels more rapidly at increasing temperatures. Principles for preparing an extracellular matrix-derived gel are provided along with preferred specific protocols for preparing gels, which are applicable and adaptable by those of skill in the art according to the needs of a particular situation and for numerous tissues including without limitation adipose or loose connective tissues.
In one embodiment, the adipose or loose connective tissue is first decellularized, leaving only the extracellular matrix, and then delipidized. In alternative embodiments, the tissue can first be delipidized, then decellularized, or the tissue can be simultaneously delipidized and decellularized. The decellularized and delipidized matrix can then be freeze-dried or lyophilized, then milled, ground or pulverized into a Fine powder, and solubilized with pepsin or other enzymes, such as, but5 not limited to, matrix metalloproteases, collagenases, and trypsin. In one embodiment, the solution can then be injected through a needle or delivered into the desired site using any delivery methods known in the art.
In one embodiment, the needle size through which the solution is injected is 25G. Dosage amounts and frequency can routinely be determined based on the varying condition of the injured tissue and patient profile. At body temperature, the solution can then form into a gel. In yet another embodiment, the composition can be delivered with cells alone or in combination with the above-described components for regenerating adipose or loose connective tissue. In yet another embodiment, the composition can be used to coat implanted devices or materials to improve adipogenesis or biocompatibility around the devices.
After the surfaces are rinsed with PBS, cells can be cultured on the adsorbed matrix. The extracellular matrix can also cause cellular differentiation of stem or progenitor cells. In some instances, a matrix resembles the in vivo adipose or loose connective tissue environment in that it contains many or all of the native chemical cues found in natural adipose or loose connective extracellular matrix. In some instances, through crosslinking or addition or other materials, the mechanical properties of healthy adult or embryonic adipose or loose connective tissue can also be mimicked.
As described herein, adipose or loose connective tissue extracellular matrix can be isolated and processed into a gel using a simple and economical process, which is amenable to scale-up for clinical translation. The cells can be any variety of cells. In some instances, the cells are a variety of adipocyte, lipoblast, or related cells including, but not limited to: stem cells, progenitors, adipocytes, lipoblasts, and fibroblasts derived from autologous or allogeneic sources. A scaffold created from adipose or loose connective extracel lular matrix is well-suited for cell transplantation in the adipose or loose connective tissue, since it more closely approximates the in vivo environment compared to currently available materials.
In addition, where proteins such as growth factors are added into the extracellular matrix, the proteins may be added into the composition, or the protein molecules may be covalently or non-covalently linked to a molecule in the matrix. The covalent linki ng of protein to matrix molecules can be accomplished by standard covalent protein linking procedures known in the art.
The protein may be covalently or linked to one or more matrix molecules. Accordingly, embryonic stem cells, fetal or adult derived stem cells, induced pluripotent stem cells, adipocyte or lipoblast progenitors, fetal and neonatal adipocytes or lipoblasts, adipose-fibroblasts, mesenchymal cells, parenchymal cells, epithelial cell s, endothelial cells, mesothelial cells, fibroblasts, hematopoietic stem cells, bone marrow-deri ved progenitor cells, skeletal cells, smooth muscle cells, macrophages, cardiocytes, myofibroblasts, and autotransplanted expanded adipocytes can be delivered by a composition herein.
In some instances, cells herein can be cultured ex vivo and in the culture dish environment differentiate directly or indirectly to adi pose or loose connective tissue cells. The cultured cell s are then transplanted into the mammal, either alone or in contact with the scaffold and other components. They may also differentiate into other lineages after introduction to organs. The adult mammal provides sources for adult stem cells, circulating endothelial precursor cell s, bone marrow-derived cells, adipose tissue, or cel ls from a specific organ.
It is known that mononuclear cells isolated from bone marrow aspirate di fferentiate i nto endothelial cells in vitro and are detected in newly formed blood vessels after intramuscular i njection. In some instances, hESC-derived adipocytes grown in the presence of a composition herein provide increased markers of maturation.
The inventive biocompatible material can be used to transplant cells, or injected alone to recruit native cells or other cytokines endogenous therapeutic agents, or act as an exogenous therapeutic agent delivery vehicle. This can be used as a research reagent for growing these cells or as a clinical reagent for culturing the cells prior to implantation.
- Extracellular Matrix Molecules.
- Product description.
- Extracellular Matrix Protocols - Second Edition | Sharona Even-Ram | Springer.
The extracellular matrix reagent can be combined with other tissue matrices and cells. Cells that can be cultured on the adsorbed matrix comprising the adipose or loose connective tissue extracellular matrix of the invention include adipocytes, lipoblasts, or other cell types relevant to adipose or loose connective ti ssue repair or regeneration, including stem cells and adipose or loose connective tissue progenitors. In some instances, a composition herein can be configured as a cell adherent.
For example, the composition herein can be coated on or mixed with a medical device or a biologic that does or does not comprise cells. Methods herein can comprise delivering the composition as a wound repair device. An injectable composition can be, without limitation, a powder, liquid, particles, fragments, gel, or emulsion. The injectable composition can be injected into a desired site comprising defective, diseased, or damaged adipose or loose connective tissue.
The compositions herein can recruit, for example without limitation, endothelial, smooth muscle, adipocyte or lipoblast progenitors, fibroblasts, and stem cells. The composition can also be delivered in a solid formulation, such as a graft or patch or associated with a cellular scaffold.
Dosages and frequency will vary depending upon the needs of the patient and judgment of the physician. A coating can be used for tissue culture applications, both research and clinical. In some instances, a coating is texturized or patterned.
go site In some instances, a method of making a coating includes adsorption or chemical linking. A thin gel or adsorbed coating can be formed using an ECM solution form of the composition. Overcoming this limitation is important for cell-mediated therapies, which rely on cultured and expanded cells retaining native cel l behavior over time. However, these approaches do not provide an accurate representation of the complex microenvironment. More complex coatings have been used, such as a combination of single proteins, and while these combinatorial signals have shown to affect cell behavior, it is not as complete as in vivo.
For a more natural matrix, cell-derived matrices can be used. While many components of extracellular matrix are similar, each tissue or organ has a unique composition, and a tissue specific naturally derived source may prove to be a better mimic of the cell microenvironment. The composition can be developed for substrate coating for a variety of applications. In some instances, the extracellular matrix of the composition retains a complex mixture of adi ose-speci fic extracel lular matrix components after solubilization. It is apparent for skilled artisans that various modifications and changes are possible and are contemplated within the scope of the current invention.
Adipose-derived mesenchymal stem cells hASCs were first isolated from the tissue according to established protocols [34, 35], Briefly, the tissue was digested in 0. The hASC-rich pellet was resuspended in mM ammonium chloride to lyse blood cells and again centrifuged at x g. After 24 hours, non-adherent cells were removed with two rinses in l x phosphate- buffered saline PBS and the remaining cells were serially passaged as hASCs.
Growth Medium was changed every days.
- Extracellular Matrix Molecules.
- Extracellular Matrices (ECM);
- Extracellular Matrix Protocols | Charles Streuli | Springer?
- Bibliographic Information?
- The Amber Spyglass (His Dark Materials, Book 3) (Deluxe 10th Anniversary Edition).
- Parallel Computing in Quantum Chemistry;
- Extracellular Matrix Protocols - Sharona Even-Ram, Vira Artym - Häftad () | Bokus;
The cells were resuspended, counted, and plated in new flasks with fresh Growth Medium. Both of these detergents have been previously shown to be effective decellularization agents . The tissue was stirred in detergent for 48 hours and subsequently thoroughly rinsed with DI water. Each group of decellularized tissue was then placed in 2. Louis, MO to remove remaining lipids. This enzymatic digestion was continued until the tissue became visibly white, approximately hours depending on the patient, or for a maximum of 72 hours if there was no change in color.
Prior to freezing, representative samples were embedded in Tissue Tek OCT compound for histological analysis. Following the decellularization and delipidization procedure, the frozen adipose-derived extracellular matrix was then lyophilized and milled using a Wiley Mini Mill. Decellularization was confirmed by staining slides with Hoechst , a fluorescent nuclear stain.
The tissue sections were fixed in acetone, rinsed, and stained in Hoechst dye at 0. The sections were then rinsed, mounted with Fluoromount Sigma-Aldrich, St. Samples of lyophilized adipose matrix were weighed and DNA was extracted according to manufacturer's specifications. As a control, lyophilized cal skin collagen Sigma-Aldrich, St. Louis, MO was included in the assay.
Louis, MO , as previously described . Sections of fresh tissue and decellularized tissue, both before and after lipase treatment, were fixed with 3. Images of the staining were taken using a Carl Zeiss Imager. The pepsin was first solubilized in 0.
The adipose matrix was digested for 48 hours at room temperature under constant stirring. Subsequently, the pH was raised to 7. Sulfated glycosaminoglycan content of the adipose matrix was quantified using a colorimetric Blyscan assay Biocolor, Carrickfergus, United Kingdom according to manufacturer's instructions.
Samples from different batches of adipose matrix were tested in triplicate and absorbance was recorded at nni using a BioTek Synergy H4 microplate reader Winooski, VT. Sections of both fresh lipoaspirate and adipose matrix were fixed with acetone and blocked with staining buffer 0. Samples were then stained with primary antibodies against collagen I, collagen I II, collagen IV, and laminin 1 : dilution. Abeam, San Francisco, CA. Among all, Pr2 and Pr4 better supported cellular viability, but Pr4 provided the best environment to promote terminal differentiation towards myocardium, endothelium and smooth muscle cell lineages.
Interestingly, we previously reported the inability of human cardiac stem cells to acquire fully differentiated state in vitro  , but d-ECM supports stem cell terminal differentiation prompting progenitor cells to further proceed in differentiation towards precursor and mature phenotype. In conclusion, the composition and architecture of scaffolds of human cardiac d-ECM depend on decellularization protocol.
We report here an adaptation of previously described protocol which yields highly preserved d-ECM, in terms of composition and architectures, that might be successfully be employed in regenerative medicine as biological scaffold. Cardiac fibroblast-derived extracellular matrix biomatrix as a model for the studies of cardiac primitive cell biological properties in normal and pathological adult human heart. Biomed Res Int. Cardiac primitive cells become committed to a cardiac fate in adult human heart with chronic ischemic disease but fail to acquire mature phenotype: genetic and phenotypic study.
Basic Res Cardiol. Presentation Type: Poster. Topic: Regenerative medicine: biomaterials for control of tissue induction. Decellularized human cardiac extracellular matrix as a natural scaffold for stem cell-based cardiac engineering.