WO2022265527A1 - A process for the decellularization of adipose tissue and use of the resulting formulation, in particular in the form of an extracellular matrix (adipoecm) - Google Patents
A process for the decellularization of adipose tissue and use of the resulting formulation, in particular in the form of an extracellular matrix (adipoecm) Download PDFInfo
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- WO2022265527A1 WO2022265527A1 PCT/PL2022/050039 PL2022050039W WO2022265527A1 WO 2022265527 A1 WO2022265527 A1 WO 2022265527A1 PL 2022050039 W PL2022050039 W PL 2022050039W WO 2022265527 A1 WO2022265527 A1 WO 2022265527A1
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- adipoecm
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- adipose tissue
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Definitions
- the invention relates to a process for the decellularization of adipose tissue and use of the resulting formulation, in particular in the form of an extracellular matrix (hereinafter referred to as “adipoECM”), in medicine and especially in plastic surgery.
- adipoECM extracellular matrix
- a common feature of classical medical biomaterials is their precisely defined composition, resulting from the use of purified ingredients.
- an lntegra®DRT biopolymer biomaterial for example, is produced, formed from porcine collagen I, sulfated glycosaminoglycans (sGAG) and a cross-linking agent (according to the specification declared by the manufacturer, Integra LifeSciences Corporation, 311 Enterprise Drive, Plainsboro, NJ 08536, USA).
- the precisely defined composition of the biomaterials enables large-scale manufacturing, and the administrative procedures related to implementation of the medical device are easier to complete.
- medical biomaterials manufactured from natural tissues with cells removed (decellularized) are different.
- a developed animal or human tissue is the starting material, from which first of all cells and, unspecifically, extracellular matrix (ECM) components are removed during preparation ⁇ ].
- ECM extracellular matrix
- an extracellular matrix specific for a particular tissue forms a consisting of the primary structural proteins and components that provide the biochemical context of the original tissue. Therefore, decellularized tissues have biological activity related to the content of structural and regulatory molecules which promote adipogenesis [2], myogenesis [3], and facilitate neurite reconstruction, for example[4].
- the resulting material cannot be so easily defined. This is applicable in particular to the components whose content in the original tissue is the lowest and whose interactions with structural elements are the weakest[5].
- the first process is based on the use of detergents (e.g. Triton X100) and nucleic acid digestion by a DNase [6,7], and the second process uses sequential trypsin digestion and isopropyl alcohol washing combined with the enzymatic breakdown of nucleic acids and lipids[8].
- detergents e.g. Triton X100
- nucleic acid digestion by a DNase [6,7] e.g. Triton X100
- isopropyl alcohol washing combined with the enzymatic breakdown of nucleic acids and lipids[8].
- Both processes can afford a decellularized adipose tissue with a DNA content of less than 50 pg/mg dry weight and without lipids (evaluated in frozen OilRed O stained sections) [7,8]. It is noted that the adipose tissue is a demanding starting material for decellularization due to the content of lipids.
- Fat is released from cells damaged during decellularization and its removal from a dense collagen network is difficult and time- consuming and typically takes about 7 days.
- an innovative process for the preparation of a decellularized adipose tissue has been developed. Compared to the aforementioned current techniques, the new process is in particular characterized by a short preparation time, i.e., no more than 48 hours, use of readily available reagents and first of all it uses a widely available iipoaspirate after plastic surgery procedures.
- the adipose tissue is a particularly readily available starting material for the preparation of decellularized biomaterials. Yellow and brown adipose tissues are known in humans[9]. The yellow adipose tissue dominates in adults and its task is to participate in sugar and fat metabolism. In addition, it is a thermal and mechanical insulator. It is found in the subcutaneous tissue, internal organs and adipose capsules of certain organs. It is formed by large (approx. 100 pm in diameter) adipocytes characterized by a single large lipid droplet which fills the entire cell. Bands of argyrophilic fibers (highly glycolyzed collagen III) are found between adipocytes.
- lobules are found between the lobules.
- Elastic fibers (elastin and fibrillin) and blood vessels are found between the lobules.
- Tissue macrophages and mesenchymal stem/stromal cells can be found around the blood vessels.
- the brown adipose tissue is characterized by smaller adipocytes (20-40 pm on average), with multiple lipid droplets inside.
- fat cells are arranged in lobules.
- multiple nerve endings can be found between the lobules. This type of tissue occurs almost exclusively in neonates, and its primary function is thermogenesis[9].
- the most important components of the adipose tissue ECM include collagen I (the primary structural component), collagen IV (forming basement membranes) as well as sulfated glycosaminoglycans (sGAGs) and hyaluronic acid which maintain adequate tissue hydration and form a site of growth factor retention (e.g. fibroblast growth factor)[9].
- collagen I the primary structural component
- collagen IV forming basement membranes
- sGAGs sulfated glycosaminoglycans
- hyaluronic acid which maintain adequate tissue hydration and form a site of growth factor retention (e.g. fibroblast growth factor)[9].
- the primary components of the extracellular matrices of various tissues are relatively well known, and the specific combination of the structural and non-structural elements of ECM forms the specific microenvironment of a tissue[10].
- tissue-specific extracellular matrix components on the fate of stem cells has been shown in a number of reports.
- Hung et al. [11] showed osteogenic properties of a 3-dimensional scaffold formed from decellularized bone and polycaprolactone.
- ASC adipose stem cells differentiated into osteocytes without the use of differentiation media (i.e DMEM with 10% serum supplemented with hydrocortisone, ascorbic acid and calcium b- glycerophosphate).
- differentiation media i.e DMEM with 10% serum supplemented with hydrocortisone, ascorbic acid and calcium b- glycerophosphate.
- the adipogenic potential of the decellularized adipose tissue matrix was reported by the group of Prof. Flynn[8]
- the alpha-amylase fraction forms as a result of decellularized adipose tissue digestion by alpha-amylase. Culturing ASC in a plastic vessel coated with the alpha-amylase fraction resulted in more rapid cell proliferation and higher expression of adipogenesis markers (such as perilipin, lipoprotein lipase) with respect to a culture on an unmodified plastic or a collagen-coated plastic.
- adipogenesis markers such as perilipin, lipoprotein lipase
- the objective of this invention is to provide a process for the relatively easy manufacture of an extracellular matrix formulation (adipoECM) through adipose tissue decellularization for use in medicine, in particular plastic surgery.
- adipoECM extracellular matrix formulation
- a particular objective is to provide a process that would shorten the procedure to approx. 2 days and also eliminate the need for using multicomponent chemical reagents and enzyme mixtures containing lipases, DNAses and RNases.
- the objective of this invention is to provide a process for the decellularization of adipose tissue and use of the resulting product, as defined in detail in the appended claims.
- IPA isopropyl alcohol (sterile reagent)
- PBS normal saline (sterile reagent)
- Water water for injection or sterile pharmaceutical water (sterile medicinal product) 0.9% NaCI: sterile medicinal product.
- the presented process can yield a decellularized adipose tissue in a much shorter time (i.e. 2 days) than competitive processes by Lauren Flynn and Stephan Badylak (7 days, for the process reported by Lauren Flynn).
- Example 1 Preparation and biochemical and mechanical characteristics of adipoECM formulations.
- adipoECM Mean DNA content per 1 mg dry weight (dw.) of adipoECM was 35.9 ⁇ 10.1 ng/mg dw. (aECMOI : 25.8 ng/mg dw., aECM02: 35.8 ng/mg dw. and aECM03: 46.1 ng/mg dw.). The result is within the reference of 50 ng/mg dw. [1] No cell nuclei were found in sections of formulations obtained from aECMOI , aECM02 and aECM03 with Masson’s staining (Fig. 1C), and no lipid droplets or cell nuclei were found in lipid and nuclear staining (Fig. 1 A and B).
- adipoECM average storage modulus (elasticity) was 307 ⁇ 76 Pa for adipoECM (aECMOI : 229.4 ⁇ 69.4, aECM02: 410.6 ⁇ 109.7 Pa and aECM03: 280.8 ⁇ 62.4) and it decreased compared to original adipose tissues of 528 ⁇ 113 Pa (AT01: 687.8 ⁇ 66.1 Pa, AT02: 439.6 ⁇ 59.4 Pa, AT03: 457.6 ⁇ 53 5 Pa) (the typical elasticity range for soft tissues is estimated at 100-1000 Pa).
- the evaluation of endotoxins and sterility of liquid collected from above adipoECM showed no growth of aerobic and anaerobic microorganisms or endotoxins as a source of pyrogens.
- Figure 1 shows results of the biochemical evaluation of an adipoECM formulation obtained using the aforementioned example embodiment of the process of the invention and the adipose tissue used. Results of OilRed O staining for lipids and hematoxylin staining for cell nuclei are shown in frozen sections of the adipose tissue (A) and aECMOI (B). The arrow shows cell nuclei, and the asterisk shows lipids.
- Masson s trichrome staining for structures, such as collagen and cell nuclei, is shown for aECMOI (C) and the result of endotoxin evaluation in the limulus amebocyte test for 3 batches: aECMOI , aECM02 and aECM03 (D).
- Example 2 Tests of the protein composition of adipoECM.
- adipoECM formulation obtained through the aforementioned example embodiment of the process of the invention was performed.
- adipoECM fragments were freeze-dried and digested with trypsin and a proteomic assay was performed (LC-MS).
- Analysis of the adipoECM composition showed major extracellular matrix proteins of the adipose tissue (Table 3), that is, collagen I and subunits forming collagen III, IV and V.
- important protein components of ECM responsible for cell interactions (laminin, fibrillin, fibronectin and tenascin) as well as those conferring tissue elasticity (elastin) were identified.
- Example 3 Proliferative activity of soft tissue stromal cells on the adipoECM matrix.
- ASC was seeded and any disruption of growth was verified.
- a metabolic cell viability test based on resazurin reduction to resorufin, which results in a second fluorescence emission maximum at 590 nm (commercial name of the assay: AlamarBlue, 10% resazurin solution), was used.
- the graph shows ASC growth in adipoECM. Proliferative activity of ASC cultured on polystyrene (2D) and on the adipoECM matrix (3D) is shown. The graph presents average results with the standard error marked. 24 hours after seeding cells into wells containing adipoECM (20x10 3 ASC/well in a 96-weel plate), adhesion to adipoECM was found for 2.5, 3.4 and 4.4 x10 3 ASC for aECMOI , aECM02 and aECM03, respectively.
- the other cells most likely underwent necrosis due to lack of space for adhesion.
- Cell growth in three-dimensional materials is slower than on polystyrene (2D).
- the cell count after 7 and 14 days was: 4.6 and 7.4 x10 3 for aECMOI , 5.4 and 6.0 x10 3 for aECM02, 8.4 and 8.6 x10 3 for aECM03.
- Example 4 Biocompatibility of adipoECM in an in vivo system.
- adipoECM prepared according to the invention was tested using immunocompetent BALB/c mice (Animalab, Poland). The idea behind the experiment was to test the response of a living organism to adipoECM at the systemic level (tests of complete blood count, biochemical components, proinflammatory cytokines and histological evaluation of the liver and spleen) and local level (evaluation of cell infiltration in the implanted biomaterials, see Fig 8).
- a market-authorized lntegra®DRT biomaterial was used, composed of the same primary components as ECM: cross-linked collagen I and sGAG.
- lntegra®DRT was selected as the control for adipoECM, because the commercially available matrix has a precisely defined biochemical composition, representing the primary components of adipoECM. Therefore, placing both biomaterials in the anatomic potential space provides an opportunity to evaluate body response and to capture biological activity which the decellularized adipose tissue may have.
- lntegra®DRT as a medical device for implantation (class 3A) is safe for living organisms.
- Figure 4 shows systemic response after 1 and 7 days based on complete blood count results.
- the complete blood count results in the mice receiving adipoECM were within the laboratory reference for BALB/c mice both on day 1 and 7.
- an elevated but still normal white blood cell count was found 1 day after the procedure, which decreased after 7 days to a mean white blood cell count.
- Complete blood counts were tested using an ABC Vet veterinary analyzer. Analysis of automated differential blood count in the lntegra®DRT group showed that increased lymphocytes (7.72 x10 3 /ml_ vs. 4.62 x10 3 /ml_ in the adipoECM group) were responsible for increased white blood cell counts. Apart from these complete blood count parameters, no statistically significant differences or exceeded reference levels were found.
- ALT alkaline phosphatase
- GPT alanine transferase
- Cre creatinine
- T-Pro total protein
- BAA urea
- Figure 5 shows systemic response after 1 and 7 days based on blood clinical chemistry tests.
- Biocompatibility of adipoECM in the in vivo system proinflammatory cytokine profile in murine plasma.
- the pro- and anti-inflammatory cytokine profile in the plasma of the mice receiving adipoECM and lntegra®DRT implants was tested 1 , 7, 30 and 60 days after the implantation of biomaterials.
- the additional timepoints, 30 and 60 days, compared to complete blood counts and blood clinical chemistry were selected considering the potential of inflammatory response to any components of the biomaterial from other species.
- Biocompatibility of adipoECM in the in vivo system histologic examinations of liver and spleen Normal tissue architectures with maintained lobules without any signs of edema, inflammatory infiltration or necrosis were found after 60 days in murine liver specimens in the control and the adipoECM groups. No fibrosis was observed eithe r. For the spleen, no differences between the control and adipoECM groups were found either: normal differentiation into cortical and medullary areas was observed, without any sites of cell infiltration or necrosis (Fig. 7).
- Figure 7 shows systemic response: histologic examinations of the liver and spleen. Paraffin sections of the spleen (A, B) and liver (C, D) were stained with hematoxylin and eosin. The structure of the spleen and liver 60 days after administration of the biomaterials was normal. The images were recorded using a Leica MIB8 light microscope. Magnification: 200x.
- Example 5 Integration of adipoECM with host tissues (simulation of augmentation).
- adipoECM To estimate the potential of adipoECM for tissue augmentation in the anatomic potential space, the ability of adipoECM to integrate with host cells away from blood cells was tested.
- lntegra®DRT as a medical device for implantation (class 3A) lntegra®DRT is safe for living organisms, but it does not contain any chemotactic agents that would promote angiogenesis and integration with host tissues except for collagen and sGAG. In addition to collagen, a decellularized adipose tissue contains other protein components that may promote angiogenesis and chemotaxis of host cells.
- Collagen matrix fibers were found in the implanted biomaterials 1 day after administering lntegra®DRT and adipoECM (blue color) (Fig. 8).
- Figure 8 presents the remodeling of the biomaterials in vivo: 1 day. Histologic preparations with lntegra®DRT (A) and adipoECM (B) 1 day after subcutaneous administration. Masson’s staining shows clearly defined biomaterials not bound with the surrounding tissues, stained blue with aniline, which confirms presence of collagen. Magnification: 200x. The images were recorded using a Leica MIB8 light microscope.
- FIG. 9 presents the remodeling of the biomaterials in vivo: 1 day, adipoECM.
- Two types of collagen fiber arrangement in adipoECM are marked with an asterisk (oblong bands) and an arrow (loose bundles). The images were recorded using a Leica MIB8 light microscope. Masson’s staining. Magnification: 400x.
- lntegra®DRT Compared to adipoECM, the structure of lntegra®DRT was regular and porous. Small, round and evenly dispersed structures stained by hematoxylin (with a diameter of about 1-2 pm) are seen throughout the cross section, most likely unspecific precipitates of the dye with negatively charged glycosaminoglycans being components of lntegra®DRT. Both lntegra®DRT and adipoECM closely adhered the surrounding tissues from the subcutaneous tissue side 60 days after administration of the biomaterials (Fig. 10A and Fig. 11 A).
- Figure 10 presents the remodeling of the biomaterials in vivo: 60 days, lntegra®DRT.
- the sections with lntegra®DRT with magnification of 200x (A) and 400x (B) 60 days after administration were subjected to Masson’s staining. No cells were found inside the material.
- the images were recorded using a Leica MIB8 light microscope.
- Figure 11 presents the remodeling of the biomaterials in vivo: 60 days, adipoECM. Histologic evaluation of adipoECM remodeling is shown with magnification of 200x (A) and 400x (B) 60 days after administration. The paraffin sections were subjected to Masson’s staining. The asterisk indicates the area with a high content of cells with loose bands of collagen fibers between cells. The arrow indicates the area with a low content of cells in which longitudinal cell nuclei can be found along compact collagen bundles. The images were recorded using a Leica MIB8 light microscope.
- adipoECM As for adipoECM, numerous host cells (Fig. 11 A) and changes in the spatial arrangement of the adipoECM components were found throughout the biomaterial cross-section, which may indicate remodeling of the decellularized adipose tissue (Fig. 11 B). Two areas can be found in the formulations: with low and high cell contents. The cells in the low content area were arranged along longitudinal compact collagen bundles. Numerous cells in high-content areas are arranged between loose collagen fiber bundles. It can be clearly seen by comparing the images of adipoECM and lntegra®DRT 60 days after their placement in the anatomic potential space in mice that only the decellularized adipose tissue was colonized and remodeled by host cells.
- adipoECM was integrated with host cells in the anatomic potential space: the material was remodeled by host cells without any signs of necrosis or inflammatory cells infiltration, and it underwent vascularization. It was thus shown that adipoECM had a potential for durable anatomic augmentation of the anatomic potential space.
- Example 6 In vivo properties of adipoECM: Identification of cells in histologic preparations.
- CD31 vascular endothelial cells
- fibroblasts fibroblasts, CD90
- CD11b tissue macrophages
- the cells that had colonized the biomaterial were to be identified in adipoECM formulations 60 days after implantation in mice. Therefore, frozen formulations were subjected to immunohisto fluoroscence staining with antibodies against CD31 (vascular endothelial cell marker), CD11b (monocyte-macrophage line marker) and CD90 (marker of mesenchymal cells, such as fibroblasts).
- CD31 vascular endothelial cell marker
- CD11b monocyte-macrophage line marker
- CD90 marker of mesenchymal cells, such as fibroblasts.
- Figure 12 presents the remodeling of the biomaterials in vivo: identification of cell populations.
- adipoECM formulations excised from mice after 60 days, single cells having the CD11b surface marker (green color, image A) and single cells having the CD90 mesenchymal cell surface marker (green color, images B) were found. Blue color in both images indicates cell nuclei. The images were recorded using a Leica MIB8 fluorescence microscope.
- CD11b- and CD90-positive cells accounted for a small minority compared to all cells with stained cell nuclei.
- Figure 12 presents the remodeling of the biomaterials in vivo: angiogenesis, adipoECM, 60 days. Sections with adipoECM subjected to trichrome Masson’s staining are shown in 400x magnification (A) and an immunohisto fluorescence preparation (B) 60 days after administration (Masson’s staining). The asterisk shows the border area between the subcutaneous tissue and adipoECM, the arrows show erythrocytes and the arrow tip indicates the largest blood vessel.
- Figure 13 presents the remodeling of the biomaterials in vivo: angiogenesis, adipoECM, 60 days. Sections with adipoECM subjected to trichrome Masson’s staining are shown in 400x magnification (A) and an immunohisto fluorescence preparation (B) 60 days after administration (Masson’s staining). The asterisk shows the border area between the subcutaneous tissue and adipoECM, the arrows show erythrocytes and the arrow tip indicates the largest blood vessel.
- Fig. 13A Blood cells are identified in trichrome preparations (Fig. 13A). Erythrocytes were found on the blood vessel cross-section. Endothelial cells were confirmed in immunohisto fluorescence preparations by detecting the CD31 marker. CD31 -positive cells were aligned peripherally and formed the blood vessel wall with a size of 48 pm x 97 pm (Fig. 13B).
- the main cell lines involved in the normal healing and remodeling of soft tissues were found.
- the presence of blood vessels and mesenchymal line cells confirms complete integration of adipoECM with host tissues.
Abstract
A process for the preparation of an extracellular matrix formulation (adipoECM) is disclosed using rapid adipose tissue decellularization to reduce the time of preparation to 2 days. The resulting formulation containing adipoECM may be used to fill volume defects of soft tissues as well as for tissue augmentation (e.g. in breast plastic surgery).
Description
A process for the decellularization of adipose tissue and use of the resulting formulation, in particular in the form of an extracellular matrix (adipoECM)
The invention relates to a process for the decellularization of adipose tissue and use of the resulting formulation, in particular in the form of an extracellular matrix (hereinafter referred to as “adipoECM”), in medicine and especially in plastic surgery.
A common feature of classical medical biomaterials is their precisely defined composition, resulting from the use of purified ingredients. In this way, an lntegra®DRT biopolymer biomaterial, for example, is produced, formed from porcine collagen I, sulfated glycosaminoglycans (sGAG) and a cross-linking agent (according to the specification declared by the manufacturer, Integra LifeSciences Corporation, 311 Enterprise Drive, Plainsboro, NJ 08536, USA). The precisely defined composition of the biomaterials enables large-scale manufacturing, and the administrative procedures related to implementation of the medical device are easier to complete. However, medical biomaterials manufactured from natural tissues with cells removed (decellularized) are different. In this case, a developed animal or human tissue is the starting material, from which first of all cells and, unspecifically, extracellular matrix (ECM) components are removed during preparation^]. As a result, an extracellular matrix specific for a particular tissue, forms a consisting of the primary structural proteins and components that provide the biochemical context of the original tissue. Therefore, decellularized tissues have biological activity related to the content of structural and regulatory molecules which promote adipogenesis [2], myogenesis [3], and facilitate neurite reconstruction, for example[4]. As a consequence of the decellularization, the resulting material cannot be so easily defined. This is applicable in particular to the components whose content in the original tissue is the lowest and whose interactions with structural elements are the weakest[5].
Two primary processes are used for the decellularization of the adipose tissue. The first process is based on the use of detergents (e.g. Triton X100) and nucleic acid digestion by a DNase [6,7], and the second process uses sequential trypsin digestion and isopropyl alcohol washing combined with the enzymatic breakdown of nucleic acids and lipids[8]. Both processes can afford a decellularized adipose tissue with a DNA content of less than 50 pg/mg dry weight and without lipids (evaluated in frozen OilRed O stained sections) [7,8].
It is noted that the adipose tissue is a demanding starting material for decellularization due to the content of lipids. Fat is released from cells damaged during decellularization and its removal from a dense collagen network is difficult and time- consuming and typically takes about 7 days. To face these challenges, an innovative process for the preparation of a decellularized adipose tissue has been developed. Compared to the aforementioned current techniques, the new process is in particular characterized by a short preparation time, i.e., no more than 48 hours, use of readily available reagents and first of all it uses a widely available iipoaspirate after plastic surgery procedures.
Among various animal or human tissues, the adipose tissue is a particularly readily available starting material for the preparation of decellularized biomaterials. Yellow and brown adipose tissues are known in humans[9]. The yellow adipose tissue dominates in adults and its task is to participate in sugar and fat metabolism. In addition, it is a thermal and mechanical insulator. It is found in the subcutaneous tissue, internal organs and adipose capsules of certain organs. It is formed by large (approx. 100 pm in diameter) adipocytes characterized by a single large lipid droplet which fills the entire cell. Bands of argyrophilic fibers (highly glycolyzed collagen III) are found between adipocytes. Together, they form lobules. Elastic fibers (elastin and fibrillin) and blood vessels are found between the lobules. Tissue macrophages and mesenchymal stem/stromal cells can be found around the blood vessels. The brown adipose tissue, in turn, is characterized by smaller adipocytes (20-40 pm on average), with multiple lipid droplets inside. As with the yellow adipose tissue, fat cells are arranged in lobules. As well as blood vessels, multiple nerve endings can be found between the lobules. This type of tissue occurs almost exclusively in neonates, and its primary function is thermogenesis[9]. The most important components of the adipose tissue ECM include collagen I (the primary structural component), collagen IV (forming basement membranes) as well as sulfated glycosaminoglycans (sGAGs) and hyaluronic acid which maintain adequate tissue hydration and form a site of growth factor retention (e.g. fibroblast growth factor)[9].
The primary components of the extracellular matrices of various tissues are relatively well known, and the specific combination of the structural and non-structural elements of ECM forms the specific microenvironment of a tissue[10].
The effect of tissue-specific extracellular matrix components on the fate of stem cells has been shown in a number of reports. Hung et al. [11] showed osteogenic
properties of a 3-dimensional scaffold formed from decellularized bone and polycaprolactone. In a construct printed based on polycaprolactone with admixed decellularized and ground trabecular bone, deposited adipose stem cells (ASC) differentiated into osteocytes without the use of differentiation media (i.e DMEM with 10% serum supplemented with hydrocortisone, ascorbic acid and calcium b- glycerophosphate).
The adipogenic potential of the decellularized adipose tissue matrix was reported by the group of Prof. Flynn[8] The alpha-amylase fraction forms as a result of decellularized adipose tissue digestion by alpha-amylase. Culturing ASC in a plastic vessel coated with the alpha-amylase fraction resulted in more rapid cell proliferation and higher expression of adipogenesis markers (such as perilipin, lipoprotein lipase) with respect to a culture on an unmodified plastic or a collagen-coated plastic.
The objective of this invention is to provide a process for the relatively easy manufacture of an extracellular matrix formulation (adipoECM) through adipose tissue decellularization for use in medicine, in particular plastic surgery. A particular objective is to provide a process that would shorten the procedure to approx. 2 days and also eliminate the need for using multicomponent chemical reagents and enzyme mixtures containing lipases, DNAses and RNases.
Surprisingly, the specific technical problem has been solved by the instant invention.
The objective of this invention is to provide a process for the decellularization of adipose tissue and use of the resulting product, as defined in detail in the appended claims.
An exemplary particularly preferred embodiment of the inventive process has been summarized in the table below.
IPA: isopropyl alcohol (sterile reagent)
PBS: normal saline (sterile reagent)
Water: water for injection or sterile pharmaceutical water (sterile medicinal product) 0.9% NaCI: sterile medicinal product.
The presented process can yield a decellularized adipose tissue in a much shorter time (i.e. 2 days) than competitive processes by Lauren Flynn and Stephan Badylak (7 days, for the process reported by Lauren Flynn).
The examples that document all aspects of this invention are provided below. The examples are included only to confirm the safety of adipoECM obtained through the rapid decellularization process and to illustrate the biological properties of adipoECM rather than as a restriction and cannot be equated to its entire scope which is defined in the claims which follow.
Example 1: Preparation and biochemical and mechanical characteristics of adipoECM formulations.
Decellularization of 3 lipoaspirates from 3 patients was performed through the rapid decellularization process of the invention, and the preferred embodiment defined below
was used, with the resulting yields as listed in Table 1 (abbreviations aECMOI , aECM02, aECM03 are used for results of respective batches, i.e. 01 , 02 and 03; the name “adipoECM” is used for product names, average results or in the general sense). Subsequently, decellularized tissue fragments were characterized biochemically (
Table 2). Mean DNA content per 1 mg dry weight (dw.) of adipoECM was 35.9±10.1 ng/mg dw. (aECMOI : 25.8 ng/mg dw., aECM02: 35.8 ng/mg dw. and aECM03: 46.1 ng/mg dw.). The result is within the reference of 50 ng/mg dw. [1] No cell nuclei were found in sections of formulations obtained from aECMOI , aECM02 and aECM03 with Masson’s staining (Fig. 1C), and no lipid droplets or cell nuclei were found in lipid and nuclear staining (Fig. 1 A and B). In addition, collagen fibers arranged in oblong bundles and fibers with a looser structure were found (Fig. 1C). The organized behavior of collagen fibers is reflected in the elasticity of adipoECM: average storage modulus (elasticity) was 307±76 Pa for adipoECM (aECMOI : 229.4±69.4, aECM02: 410.6±109.7 Pa and aECM03: 280.8±62.4) and it decreased compared to original adipose tissues of 528±113 Pa (AT01: 687.8±66.1 Pa, AT02: 439.6±59.4 Pa, AT03: 457.6±53 5 Pa) (the typical elasticity range for soft tissues is estimated at 100-1000 Pa). The evaluation of endotoxins and sterility of liquid collected from above adipoECM showed no growth of aerobic and anaerobic microorganisms or endotoxins as a source of pyrogens.
Table 2. Results of biochemical and mechanical characteristics of prepared adipoECM formulations.
dw.: dry weight, nr.: no reference.
Figure 1 shows results of the biochemical evaluation of an adipoECM formulation obtained using the aforementioned example embodiment of the process of the invention and the adipose tissue used. Results of OilRed O staining for lipids and hematoxylin staining for cell nuclei are shown in frozen sections of the adipose tissue (A) and aECMOI (B). The arrow shows cell nuclei, and the asterisk shows lipids. In addition, Masson’s trichrome staining for structures, such as collagen and cell nuclei, is shown for aECMOI (C) and the result of endotoxin evaluation in the limulus amebocyte test for 3 batches: aECMOI , aECM02 and aECM03 (D).
It is concluded to summarize the results that the process of the invention provides adipoECM with a low nucleic acid content, without any cell debris and with preserved polysaccharide components. The resulting material is sterile and free from endotoxins. Therefore, the safety of adipoECM at the biochemical quality control level has been shown.
Example 2: Tests of the protein composition of adipoECM.
Analysis of protein components of the adipoECM formulation obtained through the aforementioned example embodiment of the process of the invention was performed. adipoECM fragments were freeze-dried and digested with trypsin and a proteomic assay was performed (LC-MS). Analysis of the adipoECM composition showed major extracellular matrix proteins of the adipose tissue (Table 3), that is, collagen I and subunits forming collagen III, IV and V. In addition, important protein components of ECM responsible for cell interactions (laminin, fibrillin, fibronectin and tenascin) as well as those conferring tissue elasticity (elastin) were identified.
It was shown that the process of the invention preserved the extracellular matrix components responsible for interactions with host cells.
Example 3: Proliferative activity of soft tissue stromal cells on the adipoECM matrix.
To check if any residues of the reagents used for the preparation of adipoECM were toxic to the cells for the matrix prepared using the aforementioned example embodiment of the process of the invention, ASC was seeded and any disruption of growth was verified. A metabolic cell viability test based on resazurin reduction to resorufin, which results in a second fluorescence emission maximum at 590 nm (commercial name of the assay: AlamarBlue, 10% resazurin solution), was used.
It was found that the adipoECM formulation obtained according to the invention did not inhibit ASC growth over 14 days (Fig. 2). The graph shows ASC growth in adipoECM. Proliferative activity of ASC cultured on polystyrene (2D) and on the adipoECM matrix (3D) is shown. The graph presents average results with the standard error marked. 24 hours after seeding cells into wells containing adipoECM (20x103 ASC/well in a 96-weel plate), adhesion to adipoECM was found for 2.5, 3.4 and 4.4 x103 ASC for aECMOI , aECM02 and aECM03, respectively. The other cells most likely underwent necrosis due to lack of space for adhesion. Cell growth in three-dimensional materials is slower than on polystyrene (2D). The cell count after 7 and 14 days was: 4.6 and 7.4 x103 for aECMOI , 5.4 and 6.0 x103 for aECM02, 8.4 and 8.6 x103 for aECM03.
It was found that three independent adipoECM batches obtained by the aforementioned example embodiment of the process of the invention did not have any toxic effects against the cells and ASCs showed gradual and slow growth in their presence.
Example 4: Biocompatibility of adipoECM in an in vivo system.
To verify the biocompatibility of adipoECM prepared according to the invention in an in vivo system, tests were performed using immunocompetent BALB/c mice (Animalab, Poland). The idea behind the experiment was to test the response of a living organism to adipoECM at the systemic level (tests of complete blood count,
biochemical components, proinflammatory cytokines and histological evaluation of the liver and spleen) and local level (evaluation of cell infiltration in the implanted biomaterials, see Fig 8). As a control, a market-authorized lntegra®DRT biomaterial was used, composed of the same primary components as ECM: cross-linked collagen I and sGAG. lntegra®DRT was selected as the control for adipoECM, because the commercially available matrix has a precisely defined biochemical composition, representing the primary components of adipoECM. Therefore, placing both biomaterials in the anatomic potential space provides an opportunity to evaluate body response and to capture biological activity which the decellularized adipose tissue may have. In addition, lntegra®DRT as a medical device for implantation (class 3A) is safe for living organisms.
The in vivo experiments in BALB/c mice evaluated biocompatibility of adipoECM by comparing it to lntegra®DRT, a substitute of the connective tissue of the dermis. Compared to adipoECM, the composition of lntegra®DRT is precisely defined (collagen I, sGAG and a cross-linking agent). The respective stages of the animal experiment are shown in Figure 3 which presents the course of the in vivo experiment in mice. Images A, B and C show the procedure of administering the biomaterials (adipoECM and lntegra®DRT) through a small skin incision in the back (A), placing the biomaterial in the potential space away from wound margins (B) and placing a single suture. After a set time (1, 7, 30 and 60 days), the skin with the adhering biomaterial, e.g. aECM03 after 7 days (D), was excised and histology preparations were made.
Tests of complete blood count and blood clinical chemistry parameters were performed in the animals 1 and 7 days after the procedure. Complete blood count results showed a statistically significant difference in the white blood cell count: median white blood cell count in the group of mice with implanted lntegra®DRT was 9.9±0.4 x103/mL, and the median in the group with implanted adipoECM was 6.0±1.2 x103/mL with a reference of 14.84 x103/mL (see Fig. 4).
Figure 4 shows systemic response after 1 and 7 days based on complete blood count results. The complete blood count results in the mice receiving adipoECM were within the laboratory reference for BALB/c mice both on day 1 and 7. As for the mice which received lntegra®DRT, an elevated but still normal white blood cell count was found 1 day after the procedure, which decreased after 7 days to a mean white blood cell count. Complete blood counts were tested using an ABC Vet veterinary analyzer.
Analysis of automated differential blood count in the lntegra®DRT group showed that increased lymphocytes (7.72 x103/ml_ vs. 4.62 x103/ml_ in the adipoECM group) were responsible for increased white blood cell counts. Apart from these complete blood count parameters, no statistically significant differences or exceeded reference levels were found.
In the analysis of blood clinical chemistry parameters, no differences were found in the levels of alkaline phosphatase (ALT), alanine transferase (GPT), creatinine (Cre), total protein (T-Pro) or urea (BUA) (Fig. 5).
Figure 5 shows systemic response after 1 and 7 days based on blood clinical chemistry tests.
Analysis of clinical chemistry parameters of murine blood after administration of adipoECM and lntegra®DRT 1 and 7 days after the procedure showed no statistically significant differences in any of the tested parameters between the groups of mice receiving adipoECM and lntegra®DRT (statistical differences were verified using the Mann-Whitney test). The tests were performed using Spotchem Panel V test strips and a Spotchem EZ Chemistry analyzer.
Biocompatibility of adipoECM in the in vivo system: proinflammatory cytokine profile in murine plasma.
The pro- and anti-inflammatory cytokine profile in the plasma of the mice receiving adipoECM and lntegra®DRT implants was tested 1 , 7, 30 and 60 days after the implantation of biomaterials. The additional timepoints, 30 and 60 days, compared to complete blood counts and blood clinical chemistry were selected considering the potential of inflammatory response to any components of the biomaterial from other species. No statistically significant differences between the control group and the adipoECM group were found in any timepoint and for any of the cytokines tested (IL- 1a, IL-1P, IL-6, IL-10, IL-12p70, IL-17a, IL-23, IL-27, MCP-1 , IFN-b, IFN-g, TNF-a) (Fig. 6). Figure 6 shows Systemic response: inflammatory cytokine profile.
No statistically significant differences in the levels of the cytokines tested were found in any timepoint between the groups of mice receiving adipoECM or lntegra®DRT. Statistical analysis was performed using the Mann-Whitney test. The cytokines were assayed using the LEGENDplex Mouse Inflammation Panel 13-plex kit and a BD LSRFortessa flow cytometer.
Biocompatibility of adipoECM in the in vivo system: histologic examinations of liver and spleen
Normal tissue architectures with maintained lobules without any signs of edema, inflammatory infiltration or necrosis were found after 60 days in murine liver specimens in the control and the adipoECM groups. No fibrosis was observed eithe r. For the spleen, no differences between the control and adipoECM groups were found either: normal differentiation into cortical and medullary areas was observed, without any sites of cell infiltration or necrosis (Fig. 7).
Figure 7 shows systemic response: histologic examinations of the liver and spleen. Paraffin sections of the spleen (A, B) and liver (C, D) were stained with hematoxylin and eosin. The structure of the spleen and liver 60 days after administration of the biomaterials was normal. The images were recorded using a Leica MIB8 light microscope. Magnification: 200x.
Example 5: Integration of adipoECM with host tissues (simulation of augmentation).
To estimate the potential of adipoECM for tissue augmentation in the anatomic potential space, the ability of adipoECM to integrate with host cells away from blood cells was tested.
Tests using immunocompetent BALB/c mice were performed in an in vivo system. The idea of the experiment was to test the response of a living organism to adipoECM at systemic and local levels. As a control, a market-authorized lntegra®DRT biomaterial was used, composed of the same primary components as ECM: cross- linked collagen I and sGAG. lntegra®DRT was selected as the control for adipoECM, because the commercially available matrix has a precisely defined biochemical composition, representing the primary components of adipoECM. Therefore, placing both biomaterials in the potential space provides an opportunity to capture biological activity that underlies integration with host tissues in the anatomic potential space. lntegra®DRT as a medical device for implantation (class 3A) lntegra®DRT is safe for living organisms, but it does not contain any chemotactic agents that would promote angiogenesis and integration with host tissues except for collagen and sGAG. In addition to collagen, a decellularized adipose tissue contains other protein components that may promote angiogenesis and chemotaxis of host cells.
Collagen matrix fibers were found in the implanted biomaterials 1 day after administering lntegra®DRT and adipoECM (blue color) (Fig. 8).
Figure 8 presents the remodeling of the biomaterials in vivo: 1 day. Histologic preparations with lntegra®DRT (A) and adipoECM (B) 1 day after subcutaneous
administration. Masson’s staining shows clearly defined biomaterials not bound with the surrounding tissues, stained blue with aniline, which confirms presence of collagen. Magnification: 200x. The images were recorded using a Leica MIB8 light microscope.
The biomaterials did not contain cells and were not integrated with the surrounding tissues. Long and streaky as well as looser bundles of collagen fibers were found in the architecture of adipoECM (Fig. 9). Figure 9 presents the remodeling of the biomaterials in vivo: 1 day, adipoECM. Two types of collagen fiber arrangement in adipoECM are marked with an asterisk (oblong bands) and an arrow (loose bundles). The images were recorded using a Leica MIB8 light microscope. Masson’s staining. Magnification: 400x.
Compared to adipoECM, the structure of lntegra®DRT was regular and porous. Small, round and evenly dispersed structures stained by hematoxylin (with a diameter of about 1-2 pm) are seen throughout the cross section, most likely unspecific precipitates of the dye with negatively charged glycosaminoglycans being components of lntegra®DRT. Both lntegra®DRT and adipoECM closely adhered the surrounding tissues from the subcutaneous tissue side 60 days after administration of the biomaterials (Fig. 10A and Fig. 11 A).
Figure 10 presents the remodeling of the biomaterials in vivo: 60 days, lntegra®DRT. The sections with lntegra®DRT with magnification of 200x (A) and 400x (B) 60 days after administration were subjected to Masson’s staining. No cells were found inside the material. The images were recorded using a Leica MIB8 light microscope.
Figure 11 presents the remodeling of the biomaterials in vivo: 60 days, adipoECM. Histologic evaluation of adipoECM remodeling is shown with magnification of 200x (A) and 400x (B) 60 days after administration. The paraffin sections were subjected to Masson’s staining. The asterisk indicates the area with a high content of cells with loose bands of collagen fibers between cells. The arrow indicates the area with a low content of cells in which longitudinal cell nuclei can be found along compact collagen bundles. The images were recorded using a Leica MIB8 light microscope.
As for lntegra®DRT, cells were found only in the peripheral area closely adhering the subcutaneous tissue (Fig. 10).
As for adipoECM, numerous host cells (Fig. 11 A) and changes in the spatial arrangement of the adipoECM components were found throughout the biomaterial cross-section, which may indicate remodeling of the decellularized adipose tissue (Fig.
11 B). Two areas can be found in the formulations: with low and high cell contents. The cells in the low content area were arranged along longitudinal compact collagen bundles. Numerous cells in high-content areas are arranged between loose collagen fiber bundles. It can be clearly seen by comparing the images of adipoECM and lntegra®DRT 60 days after their placement in the anatomic potential space in mice that only the decellularized adipose tissue was colonized and remodeled by host cells.
It was found that unlike IntegraDRT, adipoECM was integrated with host cells in the anatomic potential space: the material was remodeled by host cells without any signs of necrosis or inflammatory cells infiltration, and it underwent vascularization. It was thus shown that adipoECM had a potential for durable anatomic augmentation of the anatomic potential space.
Example 6: In vivo properties of adipoECM: Identification of cells in histologic preparations.
To identify the host cells which colonized adipoECM, the primary cell lines representing vascular endothelial cells (CD31), mesenchymal line cells (fibroblasts, CD90) and tissue macrophages (CD11b) were identified.
The cells that had colonized the biomaterial were to be identified in adipoECM formulations 60 days after implantation in mice. Therefore, frozen formulations were subjected to immunohisto fluoroscence staining with antibodies against CD31 (vascular endothelial cell marker), CD11b (monocyte-macrophage line marker) and CD90 (marker of mesenchymal cells, such as fibroblasts).
Figure 12 presents the remodeling of the biomaterials in vivo: identification of cell populations. In the adipoECM formulations excised from mice after 60 days, single cells having the CD11b surface marker (green color, image A) and single cells having the CD90 mesenchymal cell surface marker (green color, images B) were found. Blue color in both images indicates cell nuclei. The images were recorded using a Leica MIB8 fluorescence microscope.
Single cells of both lines were found in the formulations stained with antibodies against tissue macrophage (CD11b) (Fig. 12A) and mesenchymal cell surface markers (CD90) (Fig. 12B). CD11b- and CD90-positive cells accounted for a small minority compared to all cells with stained cell nuclei.
Figure 12 presents the remodeling of the biomaterials in vivo: angiogenesis, adipoECM, 60 days. Sections with adipoECM subjected to trichrome Masson’s staining are shown in 400x magnification (A) and an immunohisto fluorescence preparation (B)
60 days after administration (Masson’s staining). The asterisk shows the border area between the subcutaneous tissue and adipoECM, the arrows show erythrocytes and the arrow tip indicates the largest blood vessel. B. Blue: DAPI-stained cell nuclei, red: vascular endothelial cells labeled with the antibody against the CD31 endothelial antigen. The images were recorded using a Leica MIB 8 fluorescence microscope.
Figure 13 presents the remodeling of the biomaterials in vivo: angiogenesis, adipoECM, 60 days. Sections with adipoECM subjected to trichrome Masson’s staining are shown in 400x magnification (A) and an immunohisto fluorescence preparation (B) 60 days after administration (Masson’s staining). The asterisk shows the border area between the subcutaneous tissue and adipoECM, the arrows show erythrocytes and the arrow tip indicates the largest blood vessel. B. Blue: DAPI-stained cell nuclei, red: vascular endothelial cells labeled with the antibody against the CD31 endothelial antigen. The images were recorded using a Leica MIB 8 fluorescence microscope.
Blood cells are identified in trichrome preparations (Fig. 13A). Erythrocytes were found on the blood vessel cross-section. Endothelial cells were confirmed in immunohisto fluorescence preparations by detecting the CD31 marker. CD31 -positive cells were aligned peripherally and formed the blood vessel wall with a size of 48 pm x 97 pm (Fig. 13B).
The main cell lines involved in the normal healing and remodeling of soft tissues were found. The presence of blood vessels and mesenchymal line cells confirms complete integration of adipoECM with host tissues.
References
1. Crapo PM, Gilbert TW, Badylak SF. An Overview of Tissue and Whole Organ Decellularization Processes. Biomaterials, 2011, 32: 3233-3243.
2. Turner AEB, Yu C, Bianco J, Watkins JF, Flynn LE. The Performance of Decellularized Adipose Tissue Microcarriers as an Inductive Substrate for Human Adipose-Derived Stem Cells. Biomaterials, 2012, 33 4490-4499.
3. Ungerleider JL, Johnson TD, Rao N, Christman KL. Fabrication and Characterization of Injectable Hydrogels Derived from Decellularized Skeletal and Cardiac Muscle. Methods, 2015, 15-17.
4. Medberry CJ, Crapo PM, Siu BF, Carruthers CA, Wolf MT, Nagarkar SP,
Agrawal V, Jones KE, Kelly J, Johnson SA, Velankar SS, Watkins SC, odo M, Badylak SF. Hydrogels Derived from Central Nervous System Extracellular Matrix. Biomaterials, 2013, 34: 1033-1040.
5. White LJ, Taylor AJ, Faulk DM, Keane TJ, Saldin LT, Reing JE, Swinehart IT, Turner NJ, Ratner BD, Badylak SF. The Impact of Detergents on the Tissue Decellularization Process: A ToF-SIMS Study. Acta Biomater., 2017, 50: 207- 219.
6. Wu I, Nahas Z, Kimmerling K a, Rosson GD, Elisseeff JH. An Injectable Adipose Matrix for Soft-Tissue Reconstruction. Plast. Reconstr. Surg., 2012, 129: 1247-1257.
7. Brown BN, Freund JM, Han L, Rubin JP, Reing JE, Jeffries EM, Wolf MIT, Tottey S, Barnes CA, Ratner BD, Badylak SF. Comparison of Three Methods for the Derivation of a Biologic Scaffold Composed of Adipose Tissue Extracellular Matrix. Tissue Eng. Part C Methods, 2011 , 17: 411-421 .
8. Flynn LE. The Use of Decellularized Adipose Tissue to Provide an Inductive Microenvironment for the Adipogenic Differentiation of Human Adipose -Derived Stem Cells. Biomaterials, 2010, 31: 4715-4724.
9. Cichocki T, Litwin J, Mirecka J. Kompendium Histologii: Podrpcznik Dla Studentow Nauk Medycznych i Przyrodniczych ; Wydawnictwo Uniwersytetu Jagiellonskiego: Krakow, 2016,.
10. Hynes RO. The Extracellular Matrix: Not Just Pretty Fibrils. Science, 2009, 326: 1216-1219.
11 . Hung BP, Naved BA, Nyberg EL, Dias M, Holmes CA, Elisseeff JH, Dorafshar AH, Grayson WL. Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration. ACS Biomater. Sci. Eng., 2016, 2: 1806-1816.
Materials: lntegra®DRT (CE 0086, class III).
Manufacturer: Integra LifeSciences Corporation, 311 Enterprise Drive, Plainsboro, NJ
08536, USA
Marketing Authorization Holder: Integra LifeSciences Services (France), Immeuble
Sequoia 2, 97 allee Alexandre Borodine, Parc technologique de la Porte des Alpes,
69800 Saint Priest, FRANCE
Phone: +33 (0)43747 59 50, fax: +33 (0)4 3747 5930, http://integralife.eu
Claims
1 . A process for adipose tissue decellularization, characterized in that: a) an adipose tissue sample is contacted with an aqueous solution containing gentamicin, b) the adipose tissue is digested with trypsin, c) the resulting mixture is extracted with isopropyl alcohol, d) the resulting extract is optionally sterilized and washed, and the resulting decellularized adipose tissue is separated, preferably in the form of an extracellular matrix (adipoECM) formulation.
2. A process of claim 1 , characterized in that in stage a) the adipose tissue is washed with a physiological solution containing about 100 pg/mL gentamicin, wherein an aqueous solution containing 137 mM NaCI, 2.7 mM KCI and 12 mM phosphate buffer with a pH of about 7.4 is preferably used as the physiological solution.
3. A process of claim 1 , characterized in that in stage a) the adipose tissue is washed at least twice with a physiological solution containing gentamicin, preferably washed three times at about 25°C.
4. A process of claim 1 , characterized in that in stage b) an aqueous solution containing about 0.02% (by weight) trypsin and 0.05% (by weight) sodium edetate is used.
5. A process of claim 1, characterized in that in stage b) digestion is performed for at least one hour at 37°C, while stirring the solution, preferably at about 1000 rpm.
6. A process of claim 1 , characterized in that in stage c) extraction is performed at least three times at about 30°C, while stirring the solution, preferably at about 500 rpm, wherein preferably, the first extraction is performed for at least 2 hours, the second extraction is performed for at least 4 hours, and the third extraction is performed for at least 16 hours.
7. A process of claim 1 , characterized in that in stage d) sterilization is performed in a sterile 1% aqueous peracetic acid solution containing 10% isopropyl alcohol (by
volume), for at least 2 hours at 37°C, while stirring the solution, preferably at a bout 500 rpm.
8. A process of claim 1 , characterized in that in stage d) washing with sterile water is performed, preferably for at least 30 minutes at 37°C, while stirring the solution, preferably at about 500 rpm.
9. A process of claim 1 , characterized in that in stage d) washing with a sterile 0.9% (by weight) aqueous NaCI solution is performed, preferably for at least 30 minutes at 37°C, while stirring the solution, preferably at about 500 rpm.
10. A process of claim 1 , characterized in that in stage d) washing with sterile water is performed at least twice, followed by washing with a sterile 0.9% (by weight) aqueous NaCI solution at least twice.
11. A process of claim 1 , characterized in that before starting stage a) the lipoaspirate is stored in a sterile 0.9% (by weight) NaCI solution at -25°C for at least 1 hour.
12. A process of claim 1, characterized in that before starting stage a) the tissue is thawed at room temperature, in particular about 25°C, preferably for at least 15 minutes.
13. A process of claim 1 , characterized in that before starting stage a) the size of the tissue if verified macroscopically.
14. A formulation of the decellularized adipose tissue containing the extracellular matrix (adipoECM), prepared using the process of any of the preceding claims for use in medicine, in particular in plastic surgery.
15. The formulation for use of claim 14, characterized in that it is intended for use in an injectable or solid form, in particular in minimally invasive surgery.
16. The formulation for use of claim 14, characterized in that it is intended for use in an autologous or allogenic system.
17. The formulation for use of claim 14, characterized in that it is intended for the augmentation of a human or animal body, in particular within the skin, preferably including treatment of asymmetry or defects resulting from tumor resection, injuries or pathological processes.
18. The formulation for use of claim 14, characterized in that it is intended for use in plastic surgery, in particular for breast augmentation, plastic surgery of external genital organs, face or limbs.
19. The formulation for use of claim 14, characterized in that it is intended for use in the regeneration or reconstruction of a human or animal tissue constituting a wound, in particular a fistula or skin or subcutaneous tissue loss.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110151011A1 (en) * | 2009-12-17 | 2011-06-23 | Flynn Lauren E | Decellularized Adipose Tissue |
US20180125897A1 (en) * | 2009-12-22 | 2018-05-10 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Decellularized Adipose Cell Growth Scaffold |
-
2021
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US20110151011A1 (en) * | 2009-12-17 | 2011-06-23 | Flynn Lauren E | Decellularized Adipose Tissue |
US20180125897A1 (en) * | 2009-12-22 | 2018-05-10 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Decellularized Adipose Cell Growth Scaffold |
Non-Patent Citations (2)
Title |
---|
DUNNE LINA W ET AL: "Human decellularized adipose tissue scaffold as a model for breast cancer cell growth and drug treatments", BIOMATERIALS, ELSEVIER, AMSTERDAM, NL, vol. 35, no. 18, 21 March 2014 (2014-03-21), pages 4940 - 4949, XP028841773, ISSN: 0142-9612, DOI: 10.1016/J.BIOMATERIALS.2014.03.003 * |
L.E. FLYNN: "The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells", BIOMATERIALS, vol. 31, no. 17, 1 June 2010 (2010-06-01), pages 4715 - 4724, XP055084486, ISSN: 0142-9612, DOI: 10.1016/j.biomaterials.2010.02.046 * |
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