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Last Updated: March 29, 2024

Claims for Patent: 10,017,742


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Summary for Patent: 10,017,742
Title:Process for the preparation of disinfected human cell suspensions
Abstract: In a process for preparing disinfected preparations from mammalian tissues, mammalian tissue is perfused at one step with an enzyme-free liquid antibiotic composition that includes at least one antibiotic and is contained in at least one perfusion buffer. In a step occurring after such one step, enzymatic antibiotic-free treatment of the tissue is carried out to obtain a single-cell suspension. In another step disinfected preparations are obtained.
Inventor(s): Aleksandrova; Krasimira (Hannover, DE), Barthold; Marc (Hannover, DE), Arseniev; Lubomir (Hannover, DE), Griesel; Carsten (Ladenburg, DE), Priesner; Christoph (Hannover, DE)
Assignee: Cytonet GmbH & Co. KG (Mont Saint Guibert, BE)
Application Number:14/539,158
Patent Claims:1. A process for the preparation of disinfected preparations from isolated mammalian tissues or isolated organs, comprising the following process steps: a) perfusing the isolated mammalian tissue or isolated organ with an enzyme-free liquid antibiotic composition that comprises at least one antibiotic selected from the group consisting of non-penicillin of .beta.-lactam antibiotics, aminoglycosides, glycopeptide antibiotics, and polypeptide antibiotics, and is contained in at least one perfusion buffer, b) carrying out an enzymatic, antibiotic-free treatment of the isolated mammalian tissue or the isolated organ to obtain a single-cell suspension, wherein the enzyme used for the enzymatic treatment are able to dissolve cell/cell contacts and cell/extracellular matrix contacts, and c) obtaining disinfected single-cell suspension preparations, wherein the enzymatic treatment according to step (b) takes place after step (a).

2. The process according to claim 1, wherein the tissue is liver tissue and the organ is liver.

3. The process according to claim 1, wherein the enzymatic treatment comprises a treatment with at least one of collagenase and proteinase.

4. The process according to claim 1, wherein perfusion time in process step a) is 30 to 120 min and temperature of the liquid antibiotic composition is increased during the perfusion.

5. The process according to claim 1, wherein the enzyme-free liquid antibiotic composition is contained in at least two perfusion buffers, at least one of the perfusion buffers being an EGTA- or EDTA-containing phosphate/HEPES buffer and at least one other of the buffers being a phosphate/HEPES buffer.

6. The process according to claim 1, wherein the surfaces of the tissue is rinsed with a liquid antibiotic composition prior to process step a).

7. The process according to claim 1, wherein at least one washing step using an antibiotic-free washing buffer is carried out after process step b).

8. The process according to claim 1, further comprising: washing the obtained disinfected single-cell suspension preparations with an antibiotic-free washing buffer to obtain washed disinfected single-cell suspension preparations; wherein said washing is adapted to either one of reduce or eliminate said antibiotic from said disinfected single-cell suspension so as not to have a negative effect on a microbiological sterility test.

9. The process according to claim 1, wherein the enzyme-free liquid antibiotic composition comprises Meropenem and Vancomycin.

10. The process according to claim 1, wherein the enzyme used for enzymatic treatment is a mixture of collagenase and proteinase in a ratio of enzymatic activities of 1 unit of collagenase to 40 to 60 units of proteinase.

11. A process for the preparation of disinfected preparations from isolated mammalian tissues or isolated organs, comprising the following process steps: a) perfusing the isolated mammalian tissue or isolated organ with an enzyme-free liquid antibiotic composition that comprises at least one antibiotic selected from the group consisting of non-penicillin of .beta.-lactam antibiotics, aminoglycosides, glycopeptide antibiotics, and polypeptide antibiotics, and is contained in at least one perfusion buffer, b) carrying out an enzymatic, antibiotic-free treatment of the isolated mammalian tissue or the isolated organ to obtain a single-cell suspension, wherein the enzyme used for the enzymatic treatment are able to dissolve cell/cell contacts and cell/extracellular matrix contacts, and c) obtaining disinfected single-cell suspension preparations, wherein: (i) the enzymatic treatment according to step (b) takes place after step (a), and (ii) the enzymatic treatment comprises a treatment with at least one of collagenase and proteinase, and/or (iii) perfusion time in process step a) is 30 to 120 min and temperature of the liquid antibiotic composition is increased during the perfusion, and/or (iv) the enzyme-free liquid antibiotic composition is contained in at least two perfusion buffers, at least one of the perfusion buffers being an EGTA- or EDTA-containing phosphate/HEPES buffer and at least one other of the buffers being a phosphate/HEPES buffer.

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