Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture

Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized–solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen.

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Effect of Adhesion or Collagen Molecules on Cell Attachment, Insulin Secretion, and Glucose Responsiveness in the Cultured Adult Porcine Endocrine Pancreas: A Preliminary Study

Cell Transplantation ◽

10.3727/000000003108746867 ◽

2003 ◽

Vol 12 (4) ◽

pp. 439-446 ◽

Cited By ~ 27

Author(s):

Kazuya Edamura ◽

Koko Nasu ◽

Yukiko Iwami ◽

Hiroyuki Ogawa ◽

Nobuo Sasaki ◽

...

Keyword(s):

Insulin Secretion ◽

Type I Collagen ◽

Cell Function ◽

Cell Attachment ◽

Control Group ◽

Type I ◽

Collagen Group ◽

Collagen Molecules ◽

Glucose Responsiveness

The effect of either adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness was investigated in adult porcine pancreatic endocrine (PE) cells that were cultured for a longer term in vitro. Six different types of molecules—laminin, fibronectin, poly-L-lysine (PLL), type I collagen, gelatin, and Matrigel—were used. Approximately 2.0 × 105 cells per dish of each molecule type were cultured for 4 weeks. In the laminin group, the insulin accumulation was maintained at a significantly higher level than in the control group at 4 weeks of culture, and glucose-stimulated insulin secretion and the insulin-positive rate were also higher than in the control group. In the Matrigel group, islet-like cell clusters were formed, but insulin accumulation rapidly decreased at 3–4 weeks of culture. A large number of PE cells attached tightly and spread in the fibronectin group until the fourth week of culture, but their function was not better than those in the control group. In the PLL and gelatin groups, the PE cell function was not significantly different from that of the control group. In the type I collagen group, insulin secretion was inferior to that of the other groups. The results of this study suggest that laminin is the most suitable extracellular matrix for the long-term culture preservation of PE cells.

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Molecular Dissection of the Structural Machinery Underlying the Tissue-invasive Activity of Membrane Type-1 Matrix Metalloproteinase

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0016 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3221-3233 ◽

Cited By ~ 81

Author(s):

Xiao-Yan Li ◽

Ichiro Ota ◽

Ikuo Yana ◽

Farideh Sabeh ◽

Stephen J. Weiss

Keyword(s):

Matrix Metalloproteinase ◽

Type I Collagen ◽

Cell Function ◽

Catalytic Domain ◽

Transmembrane Domain ◽

Type I ◽

Cell Trafficking ◽

Membrane Type

Membrane type-1 matrix metalloproteinase (MT1-MMP) drives cell invasion through three-dimensional (3-D) extracellular matrix (ECM) barriers dominated by type I collagen or fibrin. Based largely on analyses of its impact on cell function under two-dimensional culture conditions, MT1-MMP is categorized as a multifunctional molecule with 1) a structurally distinct, N-terminal catalytic domain; 2) a C-terminal hemopexin domain that regulates substrate recognition as well as conformation; and 3) a type I transmembrane domain whose cytosolic tail controls protease trafficking and signaling cascades. The MT1-MMP domains that subserve cell trafficking through 3-D ECM barriers in vitro or in vivo, however, remain largely undefined. Herein, we demonstrate that collagen-invasive activity is not confined strictly to the catalytic, hemopexin, transmembrane, or cytosolic domain sequences of MT1-MMP. Indeed, even a secreted collagenase supports invasion when tethered to the cell surface in the absence of the MT1-MMP hemopexin, transmembrane, and cytosolic tail domains. By contrast, the ability of MT1-MMP to support fibrin-invasive activity diverges from collagenolytic potential, and alternatively, it requires the specific participation of MT-MMP catalytic and hemopexin domains. Hence, the tissue-invasive properties of MT1-MMP are unexpectedly embedded within distinct, but parsimonious, sequences that serve to tether the requisite matrix-degradative activity to the surface of migrating cells.

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Effects of Chemotherapeutic Agents on the Function of Primary Human Osteoblast-Like Cells Derived from Children

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2003-030712 ◽

2003 ◽

Vol 88 (12) ◽

pp. 6088-6097 ◽

Cited By ~ 20

Author(s):

J. H. Davies ◽

B. A. J. Evans ◽

M. E. M. Jenney ◽

J. W. Gregory

Keyword(s):

Type I Collagen ◽

Cell Function ◽

Relative Number ◽

Chemotherapeutic Agents ◽

Type I ◽

Human Osteoblast ◽

Dihydroxyvitamin D3 ◽

Primary Human Osteoblast ◽

1,25 Dihydroxyvitamin D3

Abstract Studies in children treated with chemotherapy suggest that chemotherapeutic agents have deleterious effects on bone metabolism. We therefore evaluated the in vitro effects of clinically relevant concentrations of chemotherapeutic agents on the synthesis of type I collagen, alkaline phosphatase (AP) activity, and mineralization by primary human osteoblast-like (HOB) cells derived from children. Because serum 1,25-dihydroxyvitamin D3 concentrations may be reduced during treatment with chemotherapy, the effect of chemotherapeutic agents on HOB cells cultured in the presence or absence of 1,25-dihydroxyvitamin D3 was also evaluated. Type I collagen synthesis was reduced by all agents (P < 0.01) other than methotrexate, whereas the relative AP activity was increased (P < 0.01) by all agents. The relative number of cells staining intensely for AP after culture with agents increased (P < 0.05), and AP mRNA expression was increased (P < 0.01) with vincristine. 1,25-Dihydroxyvitamin D3 ameliorated (P < 0.01) the depletion of HOB cell numbers by chemotherapeutic agents. Furthermore, vincristine and daunorubicin inhibited 1,25-dihydroxyvitamin D3-mediated AP activity (P < 0.01). We conclude that chemotherapeutic agents can adversely affect HOB cell function, and we speculate that this observation may account, in part, for the osteopenia observed during and after treatment of children with chemotherapy.

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Collagen fibrillogenesis in vitro: interaction of types I and V collagen

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142670 ◽

1986 ◽

Vol 44 ◽

pp. 210-211

Author(s):

Arthur J. Wasserman ◽

Kathy C. Kloos ◽

David E. Birk

Keyword(s):

Type I Collagen ◽

Corneal Stroma ◽

Minor Component ◽

Homogeneous Distribution ◽

Type I ◽

Type V Collagen ◽

Fibril Morphology ◽

Type V ◽

In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

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Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta

Journal of Clinical Medicine ◽

10.3390/jcm10143141 ◽

2021 ◽

Vol 10 (14) ◽

pp. 3141

Author(s):

Hyerin Jung ◽

Yeri Alice Rim ◽

Narae Park ◽

Yoojun Nam ◽

Ji Hyeon Ju

Keyword(s):

Osteogenesis Imperfecta ◽

Type I Collagen ◽

Disease Modeling ◽

Bone Fragility ◽

Osteogenic Potential ◽

Type I ◽

Gene Correction ◽

Col1a1 Gene ◽

Collagen Formation

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

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Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays

Journal of Applied Biomaterials & Functional Materials ◽

10.1177/2280800021989698 ◽

2021 ◽

Vol 19 ◽

pp. 228080002198969

Author(s):

Min-Xia Zhang ◽

Wan-Yi Zhao ◽

Qing-Qing Fang ◽

Xiao-Feng Wang ◽

Chun-Ye Chen ◽

...

Keyword(s):

Wound Healing ◽

Wound Dressing ◽

Type I Collagen ◽

Inhibition Zone ◽

Negative Control ◽

Type I ◽

Nih3t3 Cells ◽

Post Operation

The present study was designed to fabricate a new chitosan-collagen sponge (CCS) for potential wound dressing applications. CCS was fabricated by a 3.0% chitosan mixture with a 1.0% type I collagen (7:3(w/w)) through freeze-drying. Then the dressing was prepared to evaluate its properties through a series of tests. The new-made dressing demonstrated its safety toward NIH3T3 cells. Furthermore, the CCS showed the significant surround inhibition zone than empty controls inoculated by E. coli and S. aureus. Moreover, the moisture rates of CCS were increased more rapidly than the collagen and blank sponge groups. The results revealed that the CCS had the characteristics of nontoxicity, biocompatibility, good antibacterial activity, and water retention. We used a full-thickness excisional wound healing model to evaluate the in vivo efficacy of the new dressing. The results showed remarkable healing at 14th day post-operation compared with injuries treated with collagen only as a negative control in addition to chitosan only. Our results suggest that the chitosan-collagen wound dressing were identified as a new promising candidate for further wound application.

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In vitro phosphorylation of type I collagen by cyclic AMP-dependent protein kinase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)57267-2 ◽

1986 ◽

Vol 261 (12) ◽

pp. 5674-5679

Author(s):

D B Glass ◽

J M McPherson

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Type I Collagen ◽

Type I ◽

Dependent Protein Kinase ◽

Dependent Protein

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Relative rates of biosynthesis of collagen type I, type V and type VI in calf cornea

Biochemical Journal ◽

10.1042/bj2740615 ◽

1991 ◽

Vol 274 (2) ◽

pp. 615-617 ◽

Cited By ~ 32

Author(s):

P Kern ◽

M Menasche ◽

L Robert

Keyword(s):

Type I Collagen ◽

Collagen Type ◽

Corneal Stroma ◽

Collagen Type I ◽

Type I ◽

Type Vi Collagen ◽

Sds Page ◽

Proline Incorporation ◽

Type V

The biosynthesis of type I, type V and type VI collagens was studied by incubation of calf corneas in vitro with [3H]proline as a marker. Pepsin-solubilized collagen types were isolated by salt fractionation and quantified by SDS/PAGE. Expressed as proportions of the total hydroxyproline solubilized, corneal stroma comprised 75% type I, 8% type V and 17% type VI collagen. The rates of [3H]proline incorporation, linear up to 24 h for each collagen type, were highest for type VI collagen and lowest for type I collagen. From pulse-chase experiments, the calculated apparent half-lives for types I, V and VI collagens were 36 h, 10 h and 6 h respectively.

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