EP3262399A1 - Dispositif et procédé de contrôle d'un matériau destiné à une transplantation - Google Patents

Dispositif et procédé de contrôle d'un matériau destiné à une transplantation

Info

Publication number
EP3262399A1
EP3262399A1 EP16706840.2A EP16706840A EP3262399A1 EP 3262399 A1 EP3262399 A1 EP 3262399A1 EP 16706840 A EP16706840 A EP 16706840A EP 3262399 A1 EP3262399 A1 EP 3262399A1
Authority
EP
European Patent Office
Prior art keywords
cells
raman
raman spectrum
evaluation device
electronic evaluation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP16706840.2A
Other languages
German (de)
English (en)
Inventor
Karin SCHÜTZE
Raimund SCHÜTZE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Microphotonx GmbH
Original Assignee
CELLTOOL GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CELLTOOL GmbH filed Critical CELLTOOL GmbH
Publication of EP3262399A1 publication Critical patent/EP3262399A1/fr
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures

Definitions

  • Embodiments of the invention relate to devices and methods for inspecting material for a transplant.
  • Embodiments of the invention relate in particular to devices and methods with which it can be checked whether the material is suitable for use as a transplant.
  • Skin wounds or skin diseases may require the performance of a skin graft.
  • a skin graft can be taken at a body site and transplanted to another body site.
  • Such techniques can not be satisfactory, especially if there are relatively large-area skin wounds or skin diseases.
  • Examples of such skin lesions may include burns with large and deep skin lesions, giant birthmarks or chronic wounds.
  • cultured skin can be used to treat the skin lesions.
  • Skin cells can be biopsied and applied to or embedded in a matrix. In the matrix, the skin cells can grow to the dermis and epidermis. In this way, so-called skin grafts can be bred.
  • tissue engineering T. Biedermann et al., Tissue engineering of skin for wound coverage.
  • the suitability of the material for use in skin grafting depends, for example, on the number of cells and / or on a ratio of the number of cells of different cell types. In addition, the suitability of the material for use in skin grafting may also depend on whether and to what extent cells are functionally impaired, for example, by apoptosis or necrosis.
  • Performing flow cytometry also requires that a large number of cells be pre-grown to allow for measurement. This is time-consuming and expensive (for example, with fluorescence labeling via antibodies). per-based markers). The cells are no longer available to the patient.
  • the flow cytometry provides no information about the final cell count, the ratio of the number of different cell types and / or the quality of the cells in a finished cultured graft.
  • DNA counf methods can be created on a part of the graft, but do not give any information about the cell types and / or the ratio of different cell types.
  • the material that is probed with the devices and methods may comprise cells or consist of cells.
  • the material may comprise a carrier material such as a matrix, for example a collagen matrix.
  • the devices and methods may be arranged to separately quality control the cells and the carrier material, for example the matrix.
  • the devices and methods may alternatively or additionally be adapted to quality control the cells and / or the support material, for example the matrix, in a tissue graft after culturing the cells.
  • a Raman spectroscopy is performed to examine a material.
  • One or more Raman spectra can be analyzed to see if the material is suitable for use in a transplant, using the Raman spectrum or multiple Raman spectra.
  • one or more Raman spectra may be analyzed to include a number of cells of a particular cell type in the material and / or a ratio of the number of cells of different cell types.
  • the material By evaluating one or more Raman spectra, the material can be examined objectively and quantitatively. A comparison can be made with reference spectra stored in a database to determine which cell types are present and to quantify the number of cells of one or more cell types. Alternatively or additionally, processing of the Raman spectra, for example by cluster analysis, may be performed to detect different cell types.
  • a device for testing a material for a transplant comprises a Raman spectroscopy system for performing Raman spectroscopy on the material to detect at least one Raman spectrum.
  • the device comprises an electronic evaluation device, which is set up to determine, depending on an evaluation of the at least one Raman spectrum, information on which a suitability of the material for use in the transplantation depends.
  • the material may be a material for autologous or allogeneic transplantation.
  • the material may be autologous or allogenic skin replacement.
  • the material may be an autologous or an allogenic cartilage replacement.
  • the material may be an autologous or an allogenic bone substitute
  • the material may include a skin graft.
  • the material may include artificial skin.
  • the material may comprise cultured cartilage tissue.
  • the material may comprise cultured bone tissue.
  • the material may include cells.
  • the device can be designed to prevent the cells from being applied to or inserted in a carrier material, for example a mattress. subject to Raman spectroscopy.
  • the apparatus may be arranged to automatically determine whether the cells are suitable for incorporation in a substrate, such as a matrix.
  • the device may be arranged to automatically determine which cell types are present and / or in what quantitative proportions different cell types are present.
  • the device may be configured to qualitatively or quantitatively detect contaminants and / or functional impairments of the cells from the at least one Raman spectrum.
  • the device may alternatively or additionally be arranged to subject the cells to a carrier material, for example a matrix, for Raman spectroscopy after they have been applied to or introduced into a carrier material.
  • the device may be configured to automatically determine whether the cells in the graft that comprise the cells are suitable for transplantation.
  • the device may be arranged to automatically determine which cell types are present and / or in what quantitative proportions different cell types are present.
  • the device can be set up to detect impurities and / or functional impairments of the cells in the carrier material, for example the matrix, qualitatively or quantitatively from the at least one Raman spectrum.
  • the material may comprise a carrier material, for example a matrix, into which the cells are applied or applied.
  • the matrix can be made of collagen or another material.
  • the device may be arranged to subject the carrier material, for example the matrix, to a Raman spectroscopy prior to placing or introducing the cells into a matrix.
  • the apparatus may be arranged to automatically determine whether the carrier material, for example the matrix, is suitable for introducing the cells.
  • the device may be configured to automatically determine whether the substrate, such as the matrix, is made of the desired material.
  • the device can be set up to detect impurities in the carrier material, for example the matrix, qualitatively or quantitatively from the at least one Raman spectrum.
  • the contaminants can be germs, bacteria or other foreign bodies.
  • the impurities may be contaminants of the cell population that should be present in the cultured tissue.
  • the device may alternatively or additionally be set up to subject the carrier material, for example the matrix, to Raman spectroscopy after the cells have been applied or inserted.
  • the device may be configured to automatically determine whether the carrier material, for example the matrix, in the graft comprising the matrix is suitable for transplantation.
  • the device can be set up to detect impurities and / or functional impairments of the matrix qualitatively or quantitatively from the at least one Raman spectrum.
  • the information on which the suitability of the material for use in transplantation depends can include a number of cells of a particular cell type per volume or per area.
  • the information may include the number of keratinocytes per volume or area.
  • the information may alternatively or additionally include the number of melanocytes per volume or per area.
  • the information may alternatively or additionally include the number of fibroblasts per volume or per area.
  • the information may indicate quantitatively or qualitatively whether and, if present, what impurities of the cell population are present with foreign cells.
  • the information may alternatively or additionally include the number of blood vessel cells per volume or area.
  • the information may alternatively or additionally include the number of hair follicle cells per volume or area.
  • the information may alternatively or additionally the functionality of hair follicle cells.
  • the information may alternatively or additionally include the number of corneocytes per volume or area.
  • the information may alternatively or additionally include the number of sebaceous gland cells per volume or per area.
  • the information may alternatively or additionally include the number of sweat gland cells per volume or per area.
  • the electronic evaluation device can be set up to detect, by evaluating the at least one Raman spectrum, at least one cell population of the material selected from the group consisting of: keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, Sebaceous gland cells and sweat gland cells in skin grafts
  • the electronic evaluation device can be set up to detect, by evaluating the at least one Raman spectrum, at least two different cell populations of the material, each of which is selected from the group consisting of: keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and sweat gland cells in skin grafts.
  • the electronic evaluation device can be set up to detect, by the evaluation of the at least one Raman spectrum, at least two different cell populations of the material, each of which is selected from the group consisting of: chondrocytes, chondroclasts and chondroblasts in cartilage transplants.
  • the electronic evaluation device can be set up to detect, by evaluating the at least one Raman spectrum, at least two different cell populations of the material, each of which is selected from the group consisting of: osteocytes, osteoclasts and osteoblasts in bone grafts.
  • the electronic evaluation device can be set up to detect, by evaluating the at least one Raman spectrum, at least two different cell populations of the material, each of which is selected from the group consisting of: keratinocytes, melanocytes and fibroblasts.
  • the electronic evaluation device can be set up to analyze at least two different cell types by evaluating the at least one Raman spectrum. To identify populations of the material, each of which is selected from the group consisting of: chondrocytes, chondroclasts and chondroblasts.
  • the electronic evaluation device can be set up to detect, by the evaluation of the at least one Raman spectrum, at least two different cell populations of the material, each of which is selected from the group consisting of: osteocytes, osteoclasts and osteoblasts.
  • the electronic evaluation device can be set up to determine a composition of the material in at least one area of the material.
  • the electronic evaluation device can be set up to detect the cell types present by evaluating the at least one Raman spectrum for at least one region of the material.
  • the electronic evaluation device can be set up to recognize, by evaluating the at least one Raman spectrum for at least one region of the material, in which quantitative ratio different cell types are present.
  • the different cell types may be keratinocytes, melanocytes and fibroblasts.
  • the Raman spectroscopy system may be arranged to evaluate multiple Raman spectra to perform a cluster analysis that detects relative proportions of different cell types.
  • the Raman spectroscopy system may be arranged to detect multiple Raman spectra in multiple depths of the material.
  • the electronic evaluation device can be set up to determine the composition of the material for each of the multiple depths from the respectively acquired Raman spectra.
  • the electronic evaluation device can be set up to carry out a cluster analysis of the at least one Raman spectrum.
  • the electronic evaluation device can be set up to perform a principal component analysis of the at least one Raman spectrum in order to differentiate between different cell types.
  • the electronic evaluation device can be set up to determine, depending on the cluster analysis, which proportion of keratinocytes, melanocytes, fibroblasts and / or endothelial cells is present in at least one area of the material.
  • the electronic evaluation device may comprise a memory in which information about the position of Raman peaks of different cell types of an autologous dermo-epidermal skin replacement are stored.
  • the electronic evaluation device can be set up to use a method of machine learning, in particular a method of supervised learning, in order to learn an assignment of Raman spectra and cell types.
  • the cell types may include keratinocytes, melanocytes, fibroblasts and / or endothelial cells.
  • the cell types may include keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and sweat gland cells.
  • the cell types may include chondrocytes, chondroclasts and chondroblasts.
  • the cell types may include osteocytes, osteoclasts and osteoblasts.
  • the electronic evaluation device can be set up to determine a total number of cells in the material as a function of the evaluation of the at least one Raman spectrum. For this purpose, a spectral weight of at least one Raman peak can be determined. Alternatively or additionally, a number or weight of data points determined by principal component analysis or other cluster analysis of the at least one Raman spectrum may be determined to determine the total number of cells of one or more different cell types in the material ,
  • the electronic evaluation device can be set up to carry out functional changes depending on the evaluation of the at least one Raman spectrum, in particular to recognize apoptosis and / or necrosis, at least one cell population of the material.
  • the electronic evaluation device can be set up to output information on whether the cultivated tissue is suitable for transplantation after carrying out the evaluation of several Raman spectra with which the carrier material of a cultured tissue and / or the cells of the cultured tissue were evaluated.
  • This information may be binary information that defines in a "yes or no" manner whether the cultured tissue is useful as a graft.
  • the material may include a skin graft.
  • the material may include artificial skin.
  • the artificial skin can be bred skin.
  • the artificial skin may in particular be an autologous dermo-epidermal skin replacement.
  • the artificial skin may comprise a carrier material, in particular a biodegradable carrier material, and autologous cell material.
  • a method of assaying a material for a transplant includes detecting at least one Raman spectrum of the material and determining information on which suitability of the material for use in the transplantation depends by evaluating the at least one Raman spectrum.
  • the method may be performed by the device according to one embodiment.
  • the determination of the information on which the suitability of the material for use in transplantation depends can be done automatically by an electronic computing device.
  • the material tested in the procedure may be a material for autologous transplantation.
  • the material may comprise cells.
  • the method may comprise evaluating the at least one Raman spectrum prior to introducing the cells into a support material, for example a matrix, and / or after incorporation into the support material, for example the matrix, as described in connection with the device.
  • the material may comprise a carrier material, for example a matrix.
  • the method may comprise an evaluation of the at least one Raman spectrum of the carrier material, for example the matrix, before the cells are introduced, and / or after introduction into the carrier material, for example the matrix, as described in connection with the device ,
  • the material tested in the procedure may be an autologous dermo-epidermal dermal replacement.
  • the information on which the suitability of the material for use in the transplantation depends may include a number of cells of a particular cell type per volume or area.
  • the information may include the number of keratinocytes per volume or area.
  • the information may alternatively or additionally include the number of melanocytes per volume or per area.
  • the information may alternatively or additionally include the number of fibroblasts per volume or per area.
  • the information may alternatively or additionally include the number of blood vessel cells per volume or per area. In the method, the information may alternatively or additionally include the number of hair follicle cells per volume or area. In the method, the information may alternatively or additionally include the number of hair follicle cells per volume or area. In the method, the information may alternatively or additionally include the number of hair follicle cells per volume or area. In the method, the information may alternatively or additionally include the number of corneocytes per volume or per area. In the method, the information may alternatively or additionally include the number of sebaceous gland cells per volume or per area. In the method, the information may alternatively or additionally include the number of sweat gland cells per volume or per area.
  • At least one cell population of the material selected from the group consisting of: keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and sweat gland cells can be detected.
  • At least two different cell populations of the material can be recognized, each of which is selected from the group consisting of: keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and sweat gland cells ,
  • At least two different cell populations of the material can be recognized, each of which is selected from the group consisting of: keratinocytes, melanocytes and fibroblasts.
  • At least two different cell populations of the material can be recognized, each of which is selected from the group consisting of: chondrocytes, chondroclasts and chondroblasts.
  • the electronic evaluation device can determine a composition of the material in at least one region of the material from the at least one Raman spectrum.
  • the evaluation of the at least one Raman spectrum for at least one region of the material makes it possible to identify which cell types are present.
  • By evaluating the at least one Raman spectrum for at least one region of the material it can be recognized in which quantitative ratio different cell types are present.
  • the different cell types may be keratinocytes, melanocytes and fibroblasts.
  • the method can detect multiple Raman spectra in multiple depths of material.
  • the composition of the material can be determined for each of the several depths from the respectively acquired Raman spectra.
  • the evaluation of the at least one Raman spectrum in the method may comprise performing a cluster analysis of the at least one Raman spectrum.
  • the evaluation of the at least one Raman spectrum in the method may include performing a principal component analysis of the at least one Raman spectrum to distinguish different cell types.
  • the method depending on the cluster analysis, it can be determined what proportion of keratinocytes, melanocytes, fibroblasts and / or endothelial cells are present in at least one area of the material. In the method, depending on the cluster analysis, it may determine what proportion of chondrocytes, chondroclasts, chondroblasts are present in at least a portion of the material.
  • the method may determine which portion of osteocytes, osteoclasts or osteoblasts are present in at least a portion of the material, depending on the cluster analysis.
  • the evaluation of the at least one Raman spectrum may comprise a comparison with information stored in a memory about the position of Raman peaks of different cell types of an autologous dermo-epidermal dermal substitute.
  • the evaluation of the at least one Raman spectrum may comprise a comparison with information stored in a memory about the position of Raman peaks of different cell types of an autologous cartilage replacement and / or bone replacement.
  • the method may include the implementation of a method of machine learning, in particular a method of supervised learning, by the electronic evaluation device to learn an assignment of Raman spectra and cell types.
  • the cell types may include keratinocytes, melanocytes, fibroblasts and / or endothelial cells.
  • the cell types may include keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and sweat gland cells.
  • the electronic evaluation device can determine a total number of cells in the material depending on the evaluation of the at least one Raman spectrum. For this purpose, a spectral weight of at least one Raman peak can be determined. Alternatively or additionally, in the method, a number or weight of data points determined by principal component analysis or other cluster analysis of the at least one Raman spectrum may be determined to total the number of cells of one or more different cell types in the material determine.
  • the method may include that, depending on the evaluation of the at least one Raman spectrum, functional changes, in particular apoptosis and / or necrosis, of at least one cell population of the material are detected.
  • the material tested in the procedure may include a graft.
  • the graft may comprise cultured tissue, such as cultured skin, cultured cartilage, or cultured bone tissue.
  • the material tested in the procedure may include artificial skin.
  • the artificial skin can be bred skin.
  • the artificial skin may in particular be an autologous dermo-epidermal skin replacement.
  • the artificial skin may comprise a carrier material, in particular a biodegradable carrier material, and autologous cell material.
  • the methods of embodiments may be performed remotely from the human or animal body.
  • the device of embodiments may be used for examination of the material, the examination being carried out remotely from the human or animal body.
  • the method may comprise generating the material, whereby autologous skin cells are introduced onto or into a carrier material, in particular a biodegradable matrix.
  • the methods of embodiments may be such that the recovery of the autologous skin material is not part of the claimed methods.
  • Devices and methods of embodiments allow rapid, marker-free and nondestructive testing of autologous dermo-epidermal dermal substitute or other material for its suitability for use in transplantation.
  • Figure 1 shows a schematic representation of a device according to an embodiment.
  • Figure 2 shows a material which is an autologous dermo-epidermal dermal substitute that can be tested with devices and methods of embodiments.
  • FIG. 3 shows exemplary Raman spectra which are evaluated by a device according to an exemplary embodiment.
  • FIG. 4 illustrates a processing of acquired Raman spectra by a device according to an embodiment.
  • FIG. 5 shows exemplary Raman spectra which are evaluated by a device according to an exemplary embodiment.
  • FIG. 6 shows exemplary Raman spectra which are evaluated by a device according to an exemplary embodiment.
  • FIG. 7 illustrates the spatially resolved detection of Raman spectra according to an exemplary embodiment.
  • FIG. 8 illustrates the spatially resolved detection of Raman spectra according to an exemplary embodiment.
  • FIG. 9 illustrates Raman spectra acquired at different depths of the material.
  • FIG. 10 is a flowchart of a method according to an embodiment.
  • FIG. 11 is a flowchart of a method according to an exemplary embodiment.
  • FIG. 12 is a flowchart of a method according to an embodiment.
  • FIG. 13 is a flowchart of a method according to an embodiment.
  • FIG. 14 shows exemplary Raman spectra which are detected and evaluated by a device according to an exemplary embodiment.
  • FIG. 15 illustrates processing of acquired Raman spectra by a device according to one embodiment.
  • FIG. 16 shows exemplary Raman spectra which are detected and evaluated by a device according to an exemplary embodiment.
  • FIG. 17 illustrates a processing of detected Raman spectra by a device according to an embodiment.
  • FIG. 18 shows an exemplary Raman spectra which is detected and evaluated by a device according to an exemplary embodiment.
  • Devices and methods of embodiments may be used to examine a material to determine if the material is suitable for use in a transplant.
  • Devices and methods according to exemplary embodiments can be used for the automatic checking of an autologous cultivated graft.
  • devices and methods are described in the context of techniques for testing material for use as a skin graft, the embodiments are not limited thereto.
  • Devices and methods of embodiments may generally be used to screen different types of cultured tissue for use as a graft.
  • Other examples of such tissue include cartilage or bone tissue.
  • At least one Raman spectrum of a material is detected.
  • the material can be, for example, bred skin.
  • the material may be an autologous dermo-epidermal skin substitute, in particular an autologous dermo-epidermal skin graft.
  • the material may comprise a carrier material, for example a biodegradable matrix, and autologous skin cells.
  • the at least one Raman spectrum is evaluated to obtain information on which the suitability of the material for use in skin grafting depends.
  • the information may include the number of cells of one or more cell types and / or quotients of the number of multiple cell types.
  • Figure 1 is a schematic representation of a device 1 according to an embodiment.
  • the device 1 is set up to examine a material 9 with Raman spectroscopy and to determine information on the basis of one or more detected Raman spectra, on the basis of which the material 9 is suitable for use in a skin transplantation.
  • the corresponding check of the material 9 takes place on the basis of at least one Raman spectrum which the device 1 can detect and evaluate automatically.
  • the material 9 may be mounted on a support 19 to detect the at least one Raman spectrum.
  • the device 1 comprises a Raman spectroscopy system 10 and an evaluation device 20.
  • the Raman spectroscopy system 10 is set up to detect a Raman spectrum of the material 9.
  • the material 9 may be an autologous dermo-epidermal skin replacement, in particular an autologous dermo-epidermal skin graft.
  • the recovery of autologous skin cells is not the subject of the method of embodiments.
  • the Raman spectroscopy system 10 comprises a light source 11.
  • the light source 1 1 can be a laser.
  • the laser may have a cell-sparing laser wavelength.
  • the laser wavelength can be 785 nm.
  • the light source 1 1 is arranged to output an excitation beam 17.
  • a Raman spectrometer 14 receives light 18 scattered from the material 9 by Stokes processes and / or anti-Stokes processes.
  • the Raman spectrometer 14 may comprise a diffractive element 15 and an image sensor 16 to detect the Raman spectrum of the material 9 .
  • the Raman spectroscopy system 10 may comprise further elements in a manner known per se, for example focusing optical elements 12, 13, which may be designed as lenses, and / or diaphragms.
  • the device 1 comprises an evaluation device 20.
  • the evaluation device 20 may be a computer or may comprise a computer.
  • the evaluation device 20 is coupled to the Raman spectroscopy system 10.
  • the evaluation device 20 can control the detection of the Raman spectrum by the Raman spectroscopy system 10.
  • the evaluation device 20 can control the Raman spectroscopy system 10 such that Raman spectra are detected spatially resolved at a plurality of locations of the material 9.
  • the evaluation device 20 has an interface 21 in order to receive data from the image sensor 16 of the Raman spectroscopy system 10.
  • the evaluation device has a semiconductor integrated circuit 22, which may include a processor or controller and which is configured to evaluate the detected Raman spectrum.
  • the semiconductor integrated circuit 22 is set up to determine, based on the at least one Raman spectrum, information that has an influence on whether the material 9 is already or still suitable for use in skin grafting.
  • the evaluation device 20 can output information as to whether the cultured tissue is suitable for a transplantation. This information may be binary information that defines in a "yes or no" manner whether the cultured tissue is useful as a graft.
  • the semiconductor integrated circuit 22 may be configured to detect the presence or absence of certain Raman peaks or to determine the spectral weight of Raman peaks associated with different cell types of the autologous dermo epidermal skin replacement.
  • the integrated semiconductor circuit 22 can be set up, for example, to quantitatively determine, by evaluation of the at least one Raman spectrum, whether and in what number in a volume of the material 9 keratinocytes, melanocytes, fibroblasts and / or endothelial cells are present.
  • the material 9 may also include blood and / or lymphatic vessels.
  • the integrated semiconductor circuit 22 can be set up to quantitatively determine, by evaluation of the at least one Raman spectrum, whether and in what number in a volume of the material 9 blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and / or sweat gland cells are present.
  • the semiconductor integrated circuit 22 may be configured to determine, for the material 9, local variations of Raman signals associated with different cell populations. In this way, the composition of the material 9 can be determined spatially resolved. The spatially resolved determination of the composition can be non-destructive.
  • the semiconductor integrated circuit 22 can detect different cell types, for example keratinocytes, melanocytes, fibroblasts and / or endothelial cells, based on the location of Raman peaks for the corresponding cells.
  • Information about the position and / or the spectral weight of different Raman peaks for the different cell types of an autologous dermo-epidermal dermal substitute can be stored non-volatilely in a memory of the device 1.
  • the information about the position and / or the spectral weight of different Raman peaks for the different cell types of an autologous dermis can be obtained.
  • Mo-epidermal dermal replacement can be detected by the device 1 through methods of supervised learning or other machine learning techniques
  • the semiconductor integrated circuit 22 can process acquired Raman spectra in a variety of ways, for example, statistical methods such as principal component analysis Additionally or alternatively, Raman spectra may be compared to reference data to determine which cell types are present and to determine ratios of different cell types.
  • the evaluation device 20 may include a memory 23, in which the reference data 24 are stored, which may use the integrated semiconductor circuit 22 in the evaluation of the Raman spectrum.
  • the evaluation device 20 may comprise an optical and / or acoustic output unit 25 via which, depending on the analysis of the at least one Raman spectrum, information is output which indicates whether the material 9 is suitable for use in skin grafting. Information about the total number of cells of at least one cell type and / or ratios of the number of cells of different cell types can be output.
  • FIG. 1 shows schematically a material 9, which can be examined with devices and methods according to embodiments.
  • the material 9 may be artificially bred skin.
  • the material 9 can be produced by removing skin cells and placing them in a matrix or other carrier material or be brought.
  • the matrix may comprise a hydrogel.
  • the matrix may include type 1 collagen.
  • the autologous skin cells can be increased.
  • the biopsy can be broken down into the individual cell types, multiplied and then reassembled using an extracellular framework.
  • pigment cells, blood vessels and / or lymphatic vessels may be inserted into the material 9.
  • Material 9 may or may not be autologous dermo-epidermal dermal substitute.
  • the material 9 may comprise an epidermis 31 and a dermis 32.
  • the epidermis 31 may include keratinocytes 33.
  • the epidermis may include melanocytes.
  • the dermis 32 may include fibroblasts 34. It should be understood that depending on the design of the material 9, the fibroblasts and / or various other constituents of the material 9 may be embedded in hydrogel or other matrix.
  • Figure 3 shows Raman spectra for cells of different cell types of a material 9 which is an autologous dermo-epidermal dermal substitute. Illustrated is part of a Raman spectrum 41 of keratinocytes and a Raman spectrum 42 of fibroblasts.
  • the Raman spectrum 41 of the keratinocytes and the Raman spectrum 42 of the fibroblasts differ in the position and / or the spectral weight of different Raman peaks. These differences can be used by the device 1 for the automatic differentiation of keratinocytes and fibroblasts.
  • the Raman spectrum 41 of the keratinocytes has a Raman peak 44 at a wavenumber in a wavenumber interval 47 of 1400 cm -1 to 1500 cm -1 .
  • An analysis of the Raman spectrum at wavenumber interval 47 allows a distinction between keratinocytes and fibroblasts.
  • other wave number intervals 46, 48 can be evaluated.
  • the intensity of the Raman signal for keratinocytes and fibroblasts is from 1600 cm “1 to 1670 cm -1, and the intensity of the Raman signal differs for keratinocytes and fibroblasts in a wavenumber interval 48 of 1100 cm -1 to 1390 cm -1 ,
  • Raman peaks 44,45 of a Raman spectrum can be evaluated to distinguish keratinocytes and fibroblasts. It is not necessary to compare the Raman spectrum detected on the material 9 or several Raman spectra recorded on the material 9 with information on reference spectra of keratinocytes, melanocytes, fibroblasts and / or endothelial cells.
  • the Raman spectrum or the Raman spectra can be further processed by the evaluation device 22 in order to distinguish different cell types.
  • the evaluation device 20 can, for example, carry out a cluster analysis, e.g. perform a principal component analysis of the acquired Raman spectra.
  • the result of cluster analysis can be used to distinguish keratinocytes, melanocytes, fibroblasts and / or other cells of material 9.
  • the result of the cluster analysis can be used to determine quantitative proportions of keratinocytes, melanocytes, fibroblasts and / or other cells of material 9.
  • the Raman spectrum of each cell on which the measurement is made may include a number N of intensities at different wavelengths.
  • the number N may be greater than one, in particular much greater than one.
  • each cell By comparing the spectra in each class with the spectra of already identified cells, for example from a pure culture of melanocytes or other relevant cells, each cell can be assigned a cell type.
  • the number of cells in each class can be used to quantify the proportions.
  • the number of cells in each class divided by the total number of spectra measured can then quantify the ratio of each cell type in the material.
  • FIG. 4 illustrates exemplary results of a cluster analysis performed by the evaluator 20 to determine which cell types are present in a region of the material 9.
  • the main component analysis is performed for one Raman spectrum or several Raman spectra detected on the material 9.
  • the data points are shown according to a pair of the different main components PC-1 and PC-2.
  • Figure 4 shows the data points 51 associated with keratinocytes and data points 52 associated with fibroblasts.
  • the result of the cluster analysis of the Raman spectrum acquired on the material 9 can be evaluated as to whether and how many data points lie in different regions 53, 54 of the coordinate system spanned by several principal components. For example, it can be determined how many data points lie in a region 53 that is associated with keratinocytes.
  • the evaluation device 20 can automatically determine, based on the principal component analysis of a Raman spectrum or several Raman spectra, which cell types are present and / or which portion of the cells belong to the different cell types.
  • Figure 5 shows Raman spectra for cells of different cell types of a material 9 which is an autologous dermo-epidermal dermal replacement. Illustrated as part of a Raman spectrum 61 of melanocytes and a Raman spectrum 62 of fibroblasts.
  • the Raman spectrum 61 of the melanocytes and the Raman spectrum 62 of the fibroblasts differ in the position and / or the spectral weight of different Raman peaks. These differences can be used by the device 1 for the automatic differentiation of melanocytes and fibroblasts.
  • the Raman spectrum 62 of the fibroblasts has a Raman peak at a wavenumber 63 in a wavenumber interval 66 of 1000 cm -1 to 1150 cm -1 .
  • An analysis of the Raman spectrum in wavenumber interval 66 allows a differentiation of melanocytes and fibroblasts.
  • other wavenumber intervals 67, 68 may be evaluated to distinguish melanocytes and fibroblasts from Raman peaks at different wavenumbers 64, 65.
  • the intensity of the Raman signal differs for melanocytes and fibroblasts.
  • a wavenumber interval 68 of 1575 cm -1 to 1640 cm -1 the intensity of the Raman signal differs for melanocytes and fibroblasts.
  • the device 1 can measure the ratio of the two different Raman Use peaks of measured intensities to infer the proportion of melanocytes and fibroblasts.
  • wavenumber intervals may be used to distinguish keratinocytes, melanocytes, fibroblasts, and / or other cells of material 9.
  • Figure 6 shows Raman spectra for cells of different cell types of a material 9 which is an autologous dermo-epidermal dermal substitute. Illustrated as part of a Raman spectrum 61 of melanocytes and a Raman spectrum 62 of fibroblasts.
  • the Raman spectrum 61 of the melanocytes has Raman peaks at wave numbers 73, 74 in a wavenumber interval of 2350 cm -1 to 2650 cm -1 , while the Raman spectrum 62 of the fibroblasts has no significant Raman peaks there.
  • the Raman spectrum 61 of the melanocyte Raman peaks at a wavenumber interval 75 of 2400 cm -1 to 2450 cm -1 may have a Raman peak at a wavenumber 73.
  • the Raman spectrum 61 of the melanocytes can have a Raman peak at a wavenumber interval 76 of 2500 cm -1 to 2560 cm -1 at a wavenumber of 74.
  • the Raman spectrum detected on the material 9 or for a plurality of Raman spectra acquired on the material 9 with information on reference spectra of keratinocytes, melanocytes, fibroblasts and / or endothelial cells can compare.
  • the Raman spectrum or the Raman spectra can be further processed by the evaluation device 22 in order to differentiate between different cell types.
  • the evaluation device 20 can, for example, perform a cluster analysis, for example a principal component analysis of the detected Raman spectrum.
  • the result of cluster analysis can be used to distinguish keratinocytes, melanocytes, fibroblasts and / or other cells of material 9.
  • the detection of Raman spectra can be carried out on the material 9 spatially resolved.
  • the detection can take place at a plurality of positions which are arranged on a surface of the material 9, for example on the epidermis layer.
  • Raman spectroscopy also permits detection at different depths of the material 9 without having to destroy the material 9 for this purpose.
  • the points at which one Raman spectrum or several Raman spectra are detected can define a regular or irregular lattice.
  • FIG. 7 shows a detection of Raman spectra at a plurality of regions 80. At the plurality of regions 80, which are shown as dots or filled circles 81 - 84, in each case at least one Raman spectrum can be detected. To improve the statistics, several Raman spectra can be acquired at each of the points.
  • the signal detection and relative movement between a specimen slide and optical components of the Raman spectroscopy system can be controlled automatically by the evaluation device 20.
  • Raman spectra can be detected at a plurality of separate small regions 81 -84. Although a regular arrangement of points is shown schematically in FIG. 7 at which the Raman spectra are detected, the detection can also take place on an irregular arrangement of points. Different patterns of points can be defined at which Raman spectroscopy should be performed. At least some of items 81-84 may be user definable.
  • the evaluation device 20 may include a corresponding input interface, with which a user-defined definition of those points is made possible, at each of which a Raman spectrum is to be detected.
  • the spatially resolved detection of the Raman spectra can be performed at longer intervals. According to embodiments, the spatially resolved detection of at least two Raman spectra but also on subcellular structures. At least two Raman spectra at different subcellular areas, For example, for cell nuclei and cytoplasm, detected in the material 9 and evaluated by the evaluation device 20.
  • the number of cells of one or more cell types can be quantified. For example, the keratinocytes, melanocytes and / or fibroblasts present per area can be counted.
  • FIG. 8 illustrates points 91, 92 at which the signal detection can be performed by Raman spectroscopy.
  • the signal detection by Raman spectroscopy allows the review of the material 9 at different depths 93, 94 of the material 9, without having to destroy the material 9 for this purpose.
  • the points 91, 92, at which one or more Raman spectra are detected may define a regular or irregular grid and may be arranged at different distances 93, 94 from the surface of the material 9.
  • Figure 9 shows Raman spectra 101 to 107 detected on a material 9 of fibroblasts at depths of different depths.
  • the Raman spectrum 101 was detected on the surface of a fibroblast material 9.
  • the Raman spectrum 102 was detected at a depth of 40 ⁇ m from the surface of the material 9.
  • the Raman spectrum 103 was detected at a depth of 130 ⁇ m from the surface of the material 9.
  • the Raman spectrum 104 was detected at a depth of 170 ⁇ from the surface of the material 9.
  • the Raman spectrum 105 was detected at a depth of 200 ⁇ m from the surface of the material 9.
  • the Raman spectrum 106 was detected at a depth of 220 ⁇ m from the surface of the material 9.
  • the Raman spectrum 107 was detected at a depth of 260 ⁇ from the surface of the material 9.
  • the characteristic structures of the Raman spectrum for fibroblasts can also be reliably identified at different depths 93, 94 of the material 9.
  • the determination of different cell populations ie the determination of the absolute or relative number of cells of different cell types using Raman spectroscopy, can be carried out not only on the surface but also in a three-dimensional material 9.
  • FIG. 10 is a flow chart of a method 110 according to an exemplary embodiment. The method 110 may be carried out by the device 1.
  • At step 1 1 1 at least one Raman spectrum of the material 9 is detected.
  • the light source 1 1 is controlled so that an excitation beam 17 is generated. It is also possible to record several Raman spectra. For example, several Raman spectra can be acquired at different positions of the same sample or on different samples in order to determine cell populations of different cell types.
  • the evaluation device 20 evaluates the detected Raman spectrum.
  • the evaluation device 20 can detect at least one Raman peak, which is characteristic of one of a plurality of different cell populations of the material 9.
  • the evaluation device 20 can detect at least one Raman peak that is characteristic of keratinocytes, melanocytes and / or fibroblasts.
  • the evaluation device 20 may alternatively or additionally detect at least one Raman peak that is characteristic of blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and sweat gland cells.
  • the evaluation device 20 can perform a cluster analysis on acquired Raman spectra in order to determine which cell types are present and / or in which relative number of different cell types are present.
  • FIG. 11 is a flow chart of a method 120 according to one embodiment.
  • the method 120 may be performed by the device 1.
  • the method 120 may be used to determine information concerning the usability of the material 9 for transplantation.
  • the method 120 may include detecting at least one Raman spectrum, as explained for step 11.
  • the evaluator 20 may evaluate the at least one detected Raman spectrum to determine a total number of cells of one cell type or the total number of cells of different cell types in the material 9 or a portion of the material 9. For example, the number of fibroblasts, keratinocytes and / or melanocytes in a partial volume or surface of the material 9 can be determined by evaluating the Raman spectrum. Spatially resolved, several Raman spectra can be acquired to make a count of cells of different cell types. For counting, spectral weights, intensities and / or the position of data points in a cluster analysis can be evaluated to determine the total number of cells of one cell type or the total number of cells of different cell types in the material 9 or a subregion of the material 9.
  • the evaluator 20 may determine a ratio of a number of cells of a first cell type to cells of a second cell type.
  • the evaluation device can use spectral weights, intensities and / or the position of data points in a cluster analysis in order to obtain information about the relative size of different cell populations.
  • the different cell populations may be selected from a group consisting of keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and sweat gland cells.
  • the evaluation device 20 can optionally determine from the Raman spectra whether cells of one or more cell populations undergo functional changes. conditions that reduce their suitability for transplantation. For example, the evaluation device 20 can determine whether cells of one or more cell populations, for example keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and / or sweat gland cells, are impaired in their function by apoptosis or necrosis.
  • cells of one or more cell populations for example keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and / or sweat gland cells.
  • Determination of such a functional change can be detected, for example, by shifting the data points obtained in a principal component analysis or another cluster analysis, compared to healthy, functional cells. Based on the proportion of data points that are outside the regions 53, 54 for functional cells, it can be determined whether keratinocytes, melanocytes, fibroblasts, blood vessel cells, hair follicle cells, corneocytes, sebaceous gland cells and / or sweat gland cells are subject to functional changes.
  • FIG. 12 is a flowchart of a method 130 according to one embodiment.
  • the method 130 may be performed by the device 1.
  • the method 130 may be used to determine information concerning the usability of the material 9 for transplantation.
  • the method 130 may include detecting at least one Raman spectrum, as explained for step 11.1.
  • the evaluation device 20 can evaluate the at least one detected Raman spectrum in order to determine a total number of fibroblasts in the material 9 or a subregion of the material 9. To count the number of fibroblasts, spectral weights, intensities and / or the location of data points in a cluster analysis can be evaluated to determine the total number of fibroblasts in the material 9 or a portion of the material 9.
  • the evaluator 20 may determine a ratio of a number of keratinocytes to a number of melanocytes.
  • the evaluation device can spectral weights, intensities and / or the location of data points in a Use cluster analysis to obtain information about the relative size of cell populations for keratinocytes and melanocytes.
  • Reference data, which the evaluation device 20 uses to evaluate the Raman spectra acquired at the material 9, may be stored non-volatile in the memory 23.
  • the device 1 may alternatively or additionally be adapted for machine learning techniques to learn the criteria by which different cell types can be distinguished based on Raman spectra, as described in greater detail with reference to FIG.
  • the examination of material for its suitability for transplantation can be carried out in different stages of a process for the production of autologous dermo-epidermal dermal replacement.
  • the check can be done several times sequentially.
  • the at least one Raman spectrum of cells can be detected before they are introduced or applied in a carrier material, for example a matrix.
  • a detection of different cell types a quantitative detection of relative proportions of different cell types and / or a detection of functional impairments of one or more cell types can be done.
  • the detected Raman spectrum can be compared with one or more reference spectra.
  • Analytical techniques such as cluster analysis may be used to perform detection of different cell types, quantitative detection of relative proportions of different cell types, and / or detection of functional impairments of one or more cell types.
  • a carrier material for example a matrix
  • the at least one Raman spectrum can be detected on the matrix before skin cells enter or leave skin be applied.
  • the acquired Raman spectrum can be compared with one or more reference spectra.
  • Analytical techniques such as cluster analysis, can be used to perform matrix material recognition, matrix density detection, and / or qualitative and / or quantitative contamination detection.
  • a skin replacement which is grown in a carrier material, such as a matrix, or applied cells, is suitable for transplantation.
  • a carrier material such as a matrix, or applied cells
  • different cell types can be detected. There may be a quantitative evaluation of the proportions of different cell types. It may be a recognition of functional impairments of cells of one or more different cell types.
  • the detected at least one Raman spectrum can for this purpose be compared with one or more reference spectra.
  • analysis techniques such as a cluster analysis can be used.
  • contaminations of the cell populations with foreign cells in the cultured tissue can be determined by evaluating the Raman spectra.
  • FIG. 13 is a flowchart of a method 140 according to one embodiment.
  • the method 140 may be performed by the device 1.
  • the method 140 may be used to determine information concerning the usability of the material 9 for transplantation.
  • Rules for judging the usability of the material 9 can be learned automatically by the device 1 by a method of machine learning, in particular by supervised learning.
  • the rules can be stored non-volatile in the memory 23.
  • multiple Raman spectra are acquired.
  • the multiple Raman spectra can be applied to keratinocytes, melanocytes, fibroblasts, blood vessel cells, Hair follicle cells, corneocytes, sebaceous gland cells and / or sweat gland cells are detected.
  • the multiple Raman spectra can be acquired on an autologous dermo-epidermal skin replacement of one or more patients.
  • a procedure of machine learning may be performed.
  • the procedure can be supervised learning.
  • the device 1 can receive a user input for different detected Raman spectra.
  • the user input may, for example, map Raman spectra, Raman peaks and / or clusters of a cluster analysis to different cellular constituents.
  • the device 1 may set one or more parameters of a rule set against which the device 1 evaluates Raman spectra to evaluate the suitability of the material 9 for use as a graft.
  • the evaluation device 20 can for example adapt one or more parameters of a support vector machine with which acquired Raman spectra are evaluated in order to determine information about the suitability of the material 9 for use as a transplant.
  • the learned rules for example the parameters of the support vector machine, can be stored by the device 1 in the memory 23.
  • Raman spectra may be acquired on a material 9 to be tested.
  • the stored rules may be applied to the acquired Raman spectra. This can be done, for example, as described with reference to FIG. 11 or FIG. Based on the rules, it can be determined from the acquired Raman spectra which cell types are present in the material 9. Based on the rules, it can be determined from the acquired Raman spectra in which relative number of cells of different cell types are present in the material 9. Based on the rules, it can be determined from the acquired Raman spectra whether cells of one or more cell types in the material 9 undergo functional changes compared to fully functional cells.
  • the devices and methods of embodiments can be used not only to study skin grafts but also to examine other tissues, such as cartilage and / or bone tissue, for their suitability for transplantation.
  • the evaluation device of a device can be set up to detect chondrocytes, chondroclasts and / or chondroblasts.
  • the evaluation device of a device according to one embodiment may be arranged to determine a number or density of chondrocytes, chondroclasts and / or chondroblasts in a spatially resolved manner in order to investigate the suitability of a material as a cartilaginous tissue transplant.
  • the evaluation device of a device can be designed to detect phenotypic changes of at least one cell selected from the group consisting of chondrocytes, chondroclasts and chondroblasts by evaluating the at least one Raman spectrum.
  • Figure 14 shows Raman spectra of fresh chondrocytes and chondrocytes after in vitro cultivation. A part of a Raman spectrum of fresh chondrocytes and a Raman spectrum 152 of chondrocytes after a several-day in vitro cultivation is shown as an example.
  • the Raman spectrum 151 of fresh chondrocytes and the Raman spectrum 152 of chondrocytes after several days of in vitro cultivation differ in the position and / or the spectral weight of different Raman peaks. These differences can be used by the device 1 for automatic discrimination of fresh chondrocytes and chondrocytes after several days of in vitro cultivation.
  • the Raman spectrum shows 151 fresh chondrocytes in one or more spectral regions 156, 157 has a different spectral weight than the Raman spectrum 152 of chondrocytes after a several-day in vitro cultivation.
  • Wavelength intervals 156, 157 may include one or more intervals, for example, a wavenumber interval of 900 cm -1 to 1000 cm -1 , a wavenumber interval of 950 cm -1 to 1000 cm -1 , a wavenumber interval of 1100 cm -1 to 1200 cm -1 or a wavenumber interval from 1 150 cm -1 to 1200 cm -1 .
  • Analysis of the Raman spectrum at one or more of the wavenumber intervals 156, 157 allows a decision as to whether the cultured chondrocytes are suitable for use as a graft.
  • the device 1 may use the ratio of the intensities measured at two different Raman peaks to examine cells or other material for suitability for use as a cartilage graft.
  • the Raman spectrum or the Raman spectra can be further processed by the evaluation device 22 in order to differentiate between different cell types.
  • the evaluation device 20 can, for example, perform a cluster analysis, for example a principal component analysis of the acquired Raman spectrum.
  • the result of the cluster analysis can be used to distinguish chondrocytes, chondroclasts and chondroblasts.
  • the result of cluster analysis can also be used to detect phenotypic changes in chondrocytes, chondroclasts, and chondroblasts.
  • FIG. 15 illustrates exemplary results of a cluster analysis performed by the evaluator 20 to detect phenotypic changes in chondrocytes.
  • the principal component analysis is carried out for one Raman spectrum or several Raman spectra which are generated on the material. were taken.
  • the data points are shown according to a pair of the different main components PC-1 and PC-2.
  • Figure 15 shows the data points 161 associated with fresh chondrocytes and data points 162 associated with chondrocytes having phenotypic changes.
  • the result of cluster analysis of the Raman spectrum acquired on the material can be evaluated as to whether and how many data points lie in different regions 163, 164 of the coordinate system spanned by several principal components. For example, it can be determined how many data points lie in a region 163 that is assigned to fresh chondrocytes. It can be determined how many data points lie in a region 164 associated with chondrocytes with phenotypic changes. It can be determined how many data points lie in further regions of the coordinate system spanned by a plurality of main components, which are assigned to other cellular components of the material 9, for example chondroclasts and / or chondroblasts.
  • the evaluation device 20 can automatically determine, based on the principal component analysis of a Raman spectrum or several Raman spectra, which cell types are present and / or which proportion of the cells undergoes changes.
  • Raman spectroscopy can also be used to detect disease-induced changes in cartilage cells to assess graft utility.
  • Figure 16 shows part of a Raman spectrum 171 of primary chondrocytes and part of a Raman spectrum 172 of cells derived from human chondrosarcoma cells (SW1353).
  • the Raman spectrum 171 of primary chondrocytes and the Raman spectrum 72 of cells derived from human chondrosarcoma cells (SW1353) differ in the intensity of different Raman peaks. These differences can be used by the device 1 to automatically distinguish, for example, disease-induced changes.
  • the Raman spectrum 171 of transplantable chondrocytes in one or more spectral regions 176, 177 may have a different intensity than the Raman spectrum 172 of cells undergoing disease-induced alterations.
  • the wavenumber intervals 176, 177 may include one or more intervals, for example, a wavenumber interval of 800 cm -1 to 1000 cm -1 , a wavenumber interval of 850 cm -1 to 950 cm -1 , a wavenumber interval of 1000 cm -1 to 1200 cm -1 or a wavenumber interval of 1050 cm -1 to 1 1500 cm -1 .
  • Analysis of the Raman spectrum in one or more of the wavenumber intervals 176, 177 allows a decision as to whether the cultured chondrocytes are suitable for use as a graft. It is possible, but not essential, to compare the Raman spectrum detected on the material or multiple Raman spectra acquired on the material with information about reference spectra of healthy chondrocytes, chondroclasts, and / or chondroblasts.
  • the Raman spectrum or the Raman spectra can be further processed by the evaluation device 22 in order to distinguish healthy cells from diseased cells.
  • the evaluation device 20 can, for example, perform a cluster analysis, for example a principal component analysis of the detected Raman spectrum. The result of the cluster analysis can be used to evaluate whether chondrocytes, chondroclasts and chondroblasts are subject to disease-related changes.
  • FIG. 17 illustrates exemplary results of a cluster analysis performed by the evaluation device 20 to detect disease-related changes in chondrocytes.
  • the data points are shown according to a pair of the different main components PC-1 and PC-2.
  • FIG. 17 shows data points 181 associated with chondrocytes suitable for transplantation and data points 182 associated with altered chondrocytes due to disease.
  • the result of the cluster analysis of the Raman spectrum acquired on the material can be evaluated as to whether and how many data points lie in different regions 183, 184 of the coordinate system spanned by a plurality of main components. For example, it can be determined how many data points lie in a region 183 which is assigned to chondrocytes suitable for transplantation. It can be determined how many data points lie in a region 184 that is associated with altered chondrocytes due to the disease. It can be determined how many data points lie in further regions of the coordinate system spanned by a plurality of main components, which are assigned to other cellular components of the material 9, for example chondroclasts and / or chondroblasts.
  • the evaluation device 20 can automatically determine, based on the principal component analysis of a Raman spectrum or several Raman spectra, which cell types are present and / or which fraction of the cells is subject to disease-related changes.
  • the evaluation device of a device may alternatively or additionally be set up to recognize osteocytes, osteoclasts and / or osteoblasts.
  • the evaluation device of a device may be configured to determine a number or density of osteocytes, osteoclasts and / or osteoblasts in a spatially resolved manner in order to investigate the suitability of a material as a bone graft.
  • the evaluation device of a device can be set up to examine, on the basis of the intensity and / or position of Raman peaks, whether bone tissue is suitable for a transplantation.
  • the evaluation device of a device can be set up to examine whether bone tissue is suitable for a transplantation on the basis of the intensity and / or position of Raman peaks for chemical groups in mineral and collagen phases.
  • the mineralization can be quantified.
  • Figure 18 shows part of a Raman spectrum 191 of bone tissue.
  • the Raman spectrum 191 has Raman peaks that can be assigned to mineral and collagen phases.
  • a Raman peak 192 can be identified, which can be assigned to v2 phosphate mineral.
  • the evaluation device may alternatively or additionally be set up to detect a Raman peak 193 by evaluation of the Raman spectrum 191, which can be assigned to v 4 -phosphate mineral.
  • the evaluation device may alternatively or additionally be set up to detect a Raman peak 194 by evaluation of the Raman spectrum 191, which may be assigned to the vi-phosphate mineral.
  • the evaluation device may alternatively or additionally be set up to detect one or more Raman peaks by evaluating the Raman spectrum 191, which collagen (eg amide III collagen, amide I collagen, proline ring collagen) are assigned can.
  • a relative ratio of cells selected from the group consisting of osteocytes, osteoclasts, and osteoblasts can be determined. Based on the relative intensities of one or more of these Raman peaks, disease-related changes of osteocytes, osteoclasts and / or osteoblasts can also be detected.
  • the devices and methods of embodiments may repeat the acquisition and evaluation of the Raman spectra. In this way it can be determined, for example, whether cultivated tissue has not yet reached a state in which it can be used as a graft or whether the cultivated tissue has already exceeded a state in which it can be used as a graft.
  • the devices and methods of embodiments may be used to screen a variety of different types of cultured tissue.
  • the devices and methods of embodiments may be used to determine information relevant to the utility of cultured cartilage, cultured esophageal tissue, gut tissue, or cultured gastric tissue as a graft.
  • Apparatuses and methods of embodiments may generally be used to quantitatively examine material to determine information relevant to the usability of the material 9 for transplantation.
  • the devices and methods can be used in particular for the examination of cultured transplants before they are transplanted to the patient.
  • Skin grafts are a field of application, however, the devices and methods are not limited thereto.

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Abstract

En vue du contrôle d'un matériau destiné à une transplantation, au moins un spectre Raman (41, 42) du matériau est détecté Un dispositif d'analyse électronique détermine une information dont dépend l'aptitude du matériau à être utilisé lors de transplantations, par analyse du spectre Raman (41, 42).
EP16706840.2A 2015-02-27 2016-02-24 Dispositif et procédé de contrôle d'un matériau destiné à une transplantation Pending EP3262399A1 (fr)

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DE102015203537.9A DE102015203537B3 (de) 2015-02-27 2015-02-27 Vorrichtung und Verfahren zur Überprüfung eines Materials für eine Transplantation
PCT/EP2016/053863 WO2016135194A1 (fr) 2015-02-27 2016-02-24 Dispositif et procédé de contrôle d'un matériau destiné à une transplantation

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AUPN825796A0 (en) * 1996-02-26 1996-03-14 Ashdown, Martin The application of infrared (ir) spectrometry to the investigations of components of blood and other body fluids
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WO2006113537A2 (fr) * 2005-04-14 2006-10-26 Chemimage Corporation Procede et applications pour ameliorer et imager des signaux optiques d'objets biologiques
US8253936B2 (en) * 2008-08-08 2012-08-28 Chemimage Corporation Raman characterization of transplant tissue
EP1760440A1 (fr) * 2005-08-31 2007-03-07 The Procter and Gamble Company Spectroscopie confocal Raman pour études dermatologiques
WO2007081874A2 (fr) * 2006-01-05 2007-07-19 Chemimage Corporation Systeme et methode de classification de cellules et traitement pharmaceutique de ces cellules par spectroscopie raman
DE102006053540B3 (de) 2006-11-14 2008-01-31 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Vorrichtung und Verfahren zur Analyse biologischer Proben
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US8456629B2 (en) * 2009-11-18 2013-06-04 The Regents Of The University Of California Apparatus and method for multiple-pulse impulsive stimulated raman spectroscopy
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