Muscle satellite cells are resident skeletal muscle stem cells responsible for muscle regeneration. Isolation of satellite cells is a critical process for clinical application such as drug screening and cell transplantation. Fluorescence-activated cell sorting (FACS) enables the direct isolation of satellite cells from muscle tissue. During the process used to isolate satellite cells from skeletal muscle, enzymatic digestion is the first step. Therefore, the evaluation and standardization of enzymes is important not only for reproducibility of cellular yield and viability, but also for traceability of material used in protocols.

METHODS:

The comparison of muscle digestion was performed either by a mixture of recombinant collagenase G (ColG) and collagenase H (ColH) or by a conventional collagenase II. The degree of cell damage and surface antigen expression upon collagenase treatment were analyzed by FACS. To investigate whether satellite cells isolated using recombinant collagenase can regenerate injured muscle, satellite cells were cultured, transplanted into injured muscles, and analyzed by immunostaining.

RESULTS:

We show that ColG and ColH were efficient to isolate satellite cells from mouse skeletal muscle tissue. Digestion with a combination of ColG and ColH enriched satellite cells with intact surface antigens such as α7 and β1 integrins. Furthermore, satellite cells isolated using ColG and ColH dramatically proliferated and remained undifferentiated in vitro. When transplanted, satellite cells isolated using ColG and ColH enhanced the therapeutic efficacy in vivo.

CONCLUSIONS:

Our results provide an efficient method of satellite cell preparation using recombinant collagenases with a high cell yield, viability of cells, and regeneration potency to fit the biological raw material criteria.

01 Jan 2016·Transplantation direct

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation

Article

Author: Dwulet, Francis E ; Loganathan, Gopalakrishnan ; Vargova, Lenka ; Green, Michael L ; Hughes, Michael G ; Lockridge, Amber ; Hering, Bernhard J ; Tweed, Benjamin ; Williams, Stuart K ; Wilhelm, Joshua J ; McCarthy, Robert C ; Kuriti, Manikya ; Balamurugan, Appakalai N ; Breite, Andrew G

METHODS:

We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42).

RESULTS:

Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas.

CONCLUSIONS:

A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

01 Oct 1996·Journal of clinical microbiologyQ2 · MEDICINE

Serodiagnosis of Porphyromonas gingivalis infection by immunoblot analysis with recombinant collagenase

Q2 · MEDICINE

Article

Author: Karch, H ; Schmidt, H ; Wittstock, M ; Mutters, R ; Flemmig, T F

The Porphyromonas gingivalis collagenase-specific serum immunoglobulin A (IgA), IgM, and IgG responses from 20 patients with early-onset periodontitis (EOP), 20 patients with adult periodontitis, (AP), and 20 age- and sex-matched healthy controls were examined by immunoblot analysis. A recombinant collagenase antigen used for the immunoblot analysis was produced by using the plasmid pGEX-2T, which allows the fusion between the collagenase and glutathione S-transferase. There was no significant difference in collagenase-specific IgG antibody detection between samples from the EOP, AP, and control groups. In contrast, 85% of AP and EOP sera had collagenase-specific IgA antibodies, whereas only 20% of control sera showed collagenase-specific IgA reactivity. Plaque samples from all groups were assessed by PCR with primers complementary to the collagenase-encoding gene prtC. The results indicated that 90% of AP and EOP plaque samples and 10% of control samples were positive for P. gingivalis. All patients with collagenase-specific IgA antibodies were PCR positive. The results of the study indicate a nearly complete concordance (k = 0.856) between the presence of collagenase-specific IgA antibodies and PCR detection of P. gingivalis. By using PCR as the "gold standard," the sensitivity and specificity of the IgA immunoblot test were 94.7 and 90.9%, respectively. Therefore, the recombinant collagenase is a potential candidate for use in the serodiagnosis of periodontitis.

News (Medical) associated with CNT-201

30 Jul 2024

·www.prnewswire.com

Connext Successfully Administers First Dose of CNT201 for Dupuytren's Contracture

World's First Genetically Recombinant Collagenase Therapeutics (CNT201) Begins Clinical Trials in Australia SEOUL, South Korea, July 30, 2024 /PRNewswire/ -- Connext has announced the successful first administration of CNT201, its therapeutic treatment for Dupuytren's contracture, in patients. Dupuytren's contracture is a debilitating condition characterized by the fibrosis of the fascia between the skin and tendons of the palm due to collagen buildup, causing fingers to become permanently bent and significantly impairing patients' quality of life. Currently, there is no fundamental cure for this condition. Treatments primarily involve surgical interventions like fasciectomy to alleviate symptoms, although non-surgical methods, such as localized injections of collagenase, have shown high patient satisfaction. It has received IND approval for Phase 1/2 clinical trials from the U.S. FDA and recently completed the Clinical Trial Notification (CTN) process in Australia. The ongoing clinical trial in Australia represents the Phase 1 part of the Phase 1/2 trial, conducted across three hospitals. The trial involves single administrations of four escalating dose levels in patients with Dupuytren's contracture, followed by a four-week evaluation period to assess safety, tolerability, efficacy, pharmacokinetics, and immunogenicity. Connext plans to secure proof-of-concept (PoC) data by mid-next year and determine the optimal dose for Phase 2, with the aim to commence the Phase 2 trial in the latter half of the year. Connext CEO Woojong Lee remarked, "This first administration marks a significant milestone, offering new hope to patients suffering from Dupuytren's contracture. We are dedicated to developing a safer and more effective treatment to improve their quality of life." Connext successfully raised a Series B investment at the end of last year and was selected for the Scale-Up TIPS program, enabling the initiation of global clinical trials for Dupuytren's contracture. The company is simultaneously advancing the development of additional indications, including Peyronie's disease and cellulite. Media Contact: Sangho Lee(Communication) +82-10-8829-4694 [email protected] SOURCE Connext

INDPhase 1Phase 2Gene Therapy

100 Deals associated with CNT-201

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Indication	Highest Phase	Country/Location	Organization	Date
Dupuytren Contracture	Phase 1	AU	Connext Co., Ltd.	30 Jul 2024
Cellulite	Preclinical	KR	Connext Co., Ltd.	01 Jul 2022
Lipoma	Preclinical	KR	Connext Co., Ltd.	01 Jul 2022
Penile Induration	Preclinical	KR	Connext Co., Ltd.	01 Jul 2022

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