EP-4740012-A1 - METHOD FOR EVALUATING BIOLOGICAL ACTIVITY OF BIOMATERIALS

Abstract

A method for conducting an in vitro assay of a biomaterial may include freezing a sample of biomaterial and sectioning the frozen sample of biomaterial into at least one section of biomaterial. The at least one section of biomaterial may have a thickness of about 10 μm to about 50 μm. The method may further include placing the at least one section of biomaterial onto a slide; affixing a neuron or a group of neurons to one end portion of the at least one section of biomaterial to create a test construct; incubating the test construct; and determining an amount of neurite growth from the neuron or the group of neurons along the at least one section of biomaterial.

Inventors

• ALSMADI, Nesreen
• TAJDARAN, Kasra

Assignees

• AxoGen Corporation

Dates

Publication Date

20260513

Application Date

20240821

Claims (20)

1. 1 . A method for conducting an in vitro assay of a biomaterial, the method comprising: freezing a sample of biomaterial; sectioning the frozen sample of biomaterial into at least one section of biomaterial, wherein the at least one section of biomaterial has a thickness of about 10 pm to about 50 pm; placing the at least one section of biomaterial onto a slide; affixing a neuron or a group of neurons to one end portion of the at least one section of biomaterial to create a test construct; incubating the test construct; and determining an amount of neurite growth from the neuron or the group of neurons along the at least one section of biomaterial.
2. 2. The method according to claim 1 , wherein determining the amount of neurite growth comprises measuring at least one longest outgrowing neurite from the neuron or the group of neurons.
3. 3. The method according to claim 2, wherein measuring the at least one longest outgrowing neurite from the neuron or the group of neurons comprises measuring a subset of the longest outgrowing neurites and averaging the lengths of the subset of the longest outgrowing neurites.
4. 4. The method according to claim 1 , wherein the neuron or each neuron, of the group of neurons, is a dorsal root ganglia (DRG).
5. 5. The method according to claim 1 , further comprising: staining the test construct prior to determining the amount of neurite growth from the neuron or group of neurons.
6. 6. The method according to claim 5, wherein incubating the plurality of sections of biomaterial includes exposing the test construct to carbon dioxide.
7. 7. The method according to claim 1 , further comprising using a cryofreezing media when freezing the biomaterial.
8. 8. The method according to claim 1 , the slide has a positive charge on a surface thereof on which the at least one section is placed.
9. 9. The method according to claim 1 , further comprising thawing the at least one section of biomaterial and washing the at least one section of biomaterial with a buffer solution prior to affixing the neuron or the group of neurons.
10. 10. The method according to claim 1 , wherein the at least one section of biomaterial comprises a plurality of sections of biomaterial, and wherein each of the plurality of sections of biomaterial is placed onto a respective slide.
11. 11 . The method according to claim 10, further comprising treating a first subset of the plurality of sections of biomaterial with an agent, and further comprising determining an effect of the agent on neurite growth by comparing an amount of neurite growth in the treated first subset of sections of biomaterial to an amount of neurite growth in a second subset of untreated sections of the plurality of sections of biomaterial.
12. 12. The method according to claim 1 , wherein the biomaterial is a nerve graft.
13. 13. A method for conducting an in vitro assay of a biomaterial, the method comprising: freezing a sample of biomaterial using a cryofreezing media; sectioning the frozen sample of biomaterial into a plurality of sections of biomaterial, each section having a thickness of about 10 pm to about 50 pm; placing each section, of the plurality of sections of biomaterial, onto a slide, of a plurality of slides, wherein each slide, of the plurality of slides, has a positive charge on a surface thereof on which a section, of the plurality of sections of biomaterial, is placed; thawing the plurality of sections of biomaterial; washing the plurality of sections of biomaterial; affixing a neuron or a group of neurons to one end portion of each of the plurality of sections of biomaterial; incubating the plurality of sections of biomaterial, with the neuron or the group of neurons affixed thereto; staining the plurality of incubated sections of biomaterial; and determining, for each of the plurality of incubated sections of biomaterial, an amount of neurite growth from the neuron or the group of neurons along the section of biomaterial.
14. 14. The method according to claim 13, wherein determining, for each of the plurality of incubated sections of biomaterial, the amount of neurite growth comprises measuring at least one longest outgrowing neurite from the neuron or the group of neurons.
15. 15. The method according to claim 13, wherein the neuron or each neuron, of the group of neurons, is a dorsal root ganglia (DRG), and wherein the biomaterial is a nerve graft.
16. 16. The method according to claim 13, further comprising freezing the plurality of sections of biomaterial on the plurality of slides prior to thawing the plurality of sections of biomaterial.
17. 17. The method according to claim 13, wherein the plurality of slides are frosted or coated with a coating material.
18. 18. The method according to claim 13, further comprising treating a first subset of the plurality of sections of biomaterial with an agent, and further comprising determining an effect of the agent on neurite growth by comparing an amount of neurite growth in the treated first subset of sections of biomaterial to an amount of neurite growth in a second subset of untreated sections of the plurality of sections of biomaterial.
19. 19. The method according to claim 13, wherein incubating the plurality of sections of biomaterial occurs at a temperature of less than 40°C for about 3 days to about 10 days.
20. 20. A method for conducting an in vitro assay of nerve tissue, the method comprising: freezing a sample of nerve tissue using; sectioning the frozen sample of nerve tissue into a plurality of sections of nerve tissue, each section having a thickness of about 10 pm to about 50 pm; placing each section, of the plurality of sections of nerve tissue, onto a positively charged slide, of a plurality of slides; thawing the plurality of sections of nerve tissue; washing the plurality of sections of nerve tissue; affixing a neuron or a group of neurons to one end portion of each of the plurality of sections of nerve tissue; incubating the plurality of sections of nerve tissue, with the neuron or the group of neurons affixed thereto, to allow for neurite growth from the neuron or the group of neurons along the nerve tissue; staining the plurality of incubated sections of nerve tissue; and measuring, on each slide of the plurality of slides, at least one longest outgrowing neurite from the neuron or the group of neurons along the nerve tissue.

Description

METHOD FOR EVALUATING BIOLOGICAL ACTIVITY OF BIOMATERIALS CROSS-REFERENCE TO RELATED APPLICATION [0001] This patent application claims the benefit of priority to U.S. NonProvisional Patent Application No. 18/808,470, filed August 19, 2024, which claims the benefit of priority to U.S. Provisional Patent Application No. 63/535,685, filed August 31 , 2023, the entireties of which are incorporated herein by reference. TECHNICAL FIELD [0002] Various embodiments of the present disclosure relate generally to a method for evaluating biological activity of biological materials, and, more particularly, to an in vitro bioassay platform to evaluate potency of biological materials. BACKGROUND [0003] To determine the suitability of a tissue graft for surgical repair of damaged or severed tissue, in vitro bioassays may be performed to evaluate potency of a tissue graft, e.g., its ability to facilitate regrowth. Known assays may require more time and cost to conduct. For example, such assays may include affixing a tissue to be regenerated to a three-dimensional, full-thickness graft to form a test construct, culturing the test construct in a medium for a period of time to allow growth of the tissue into the graft, and then sectioning the test construct to attempt to evaluate the extent of the growth into the three-dimensional graft, since growth within the graft cannot be visually assessed without sectioning. The sections may then need to be stained in order to observe the components of the sections. Further, taking sections of the tissue graft may be an imperfect measure, because the exact extent of the tissue growth within the graft may be difficult to assess and may occur between sections. The sections must be sorted through to identify the longest outgrowth of tissue and quantify the growth. Additionally, the neurite growth does not occur in a straight line, making it challenging to accurately track the extent of the growth. [0004] There is a need, however, for a method for evaluating the suitability of a biological material for use in surgical repair that requires less biological material and less time to complete an evaluation. In addition, there is a need for a method in which sections of the biological material are better suited for depositing on a slide used during the analysis, and a method in which analysis does not require sorting through cross-sectional slices to assess the longest outgrowth, and which allows for improved measurement of tissue growth. [0005] The present invention is directed to overcoming one or more of these above-referenced challenges. SUMMARY OF THE INVENTION [0006] A method for conducting an in vitro assay of a biomaterial may include freezing a sample of biomaterial and sectioning the frozen sample of biomaterial into at least one section of biomaterial. The at least one section of biomaterial may have a thickness of about 10 pm to about 50 pm. The method may further include placing the at least one section of biomaterial onto a slide; affixing a neuron or a group of neurons to one end portion of the at least one section of biomaterial to create a test construct; incubating the test construct; and determining an amount of neurite growth from the neuron or the group of neurons along the at least one section of biomaterial. [0007] Determining the amount of neurite growth may include measuring at least one longest outgrowing neurite from the neuron or the group of neurons; measuring the at least one longest outgrowing neurite from the neuron or the group of neurons may include measuring a subset of the longest outgrowing neurites and averaging the lengths of the subset of the longest outgrowing neurites; the neuron or each neuron, of the group of neurons, may be a dorsal root ganglia (DRG); the method may further include staining the test construct prior to determining the amount of neurite growth from the neuron or group of neurons; incubating the plurality of sections of biomaterial may include exposing the test construct to carbon dioxide; the method may include using a cryofreezing media when freezing the biomaterial; the slide may have a positive charge on a surface thereof on which the at least one section is placed; the method may include thawing the at least one section of biomaterial and washing the at least one section of biomaterial with a buffer solution prior to affixing the neuron or the group of neurons; the at least one section of biomaterial may include a plurality of sections of biomaterial, and each of the plurality of sections of biomaterial may be placed onto a respective slide; the method may include treating a first subset of the plurality of sections of biomaterial with an agent, and may further comprise determining an effect of the agent on neurite growth by comparing an amount of neurite growth in the treated first subset of sections of biomaterial to an amount of neurite growth in a second subset of untreated sections of the plurality of sections of biomaterial