EP2430155A1 - Séparation automatisée de couches tissulaires - Google Patents

Séparation automatisée de couches tissulaires

Info

Publication number
EP2430155A1
EP2430155A1 EP10706522A EP10706522A EP2430155A1 EP 2430155 A1 EP2430155 A1 EP 2430155A1 EP 10706522 A EP10706522 A EP 10706522A EP 10706522 A EP10706522 A EP 10706522A EP 2430155 A1 EP2430155 A1 EP 2430155A1
Authority
EP
European Patent Office
Prior art keywords
tissue
layer
cluster
blade
epithelial cell
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.)
Withdrawn
Application number
EP10706522A
Other languages
German (de)
English (en)
Inventor
Frank Pretzsch
Ulrike Koropp
Andrea Heymer
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.)
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Original Assignee
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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 Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV filed Critical Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Publication of EP2430155A1 publication Critical patent/EP2430155A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin

Definitions

  • the invention relates to a method for automatically separating and isolating tissue cells of a tissue layer from a multilayered biological tissue of the animal or human body, particularly epithelial cells from skin biopsates, as well as to a device for automatically carrying out the separation.
  • the invention relates to the technical field of tissue engineering.
  • the principle of tissue engineering consists substantially in isolating vital cells or cell clusters from biological tissue which can be recovered in a separate method sequence from the human or animal body in the form of donor tissue as what is known as a biopsate and in constituting new tissues therefrom.
  • the isolated cells are propagated and subsequently applied in order to build up newly constituted three- dimensional tissue structures, for example newly constituted skin equivalents.
  • Newly constituted tissues of this type can then be used as test systems in research, in particular for researching active ingredients or as transplants in medicine, in order to replace lost organ functions.
  • two-layered skin models are preferably used as test systems for active ingredients, chemicals and cosmetics and provide an alternative to animal testing.
  • fibroblasts and keratinocytes which, as is known, are isolated from a multilayered skin tissue, and in particular from prepuce biopsates.
  • the epithelial layer In the manual methods, in order to recover individual epithelial cells from the manually separated epithelial layer, the epithelial layer must firstly be homogenised in a further method step and subsequently be subjected to a trypsin treatment. This requires a long processing time in order to recover a large number of singled-out epithelial cells.
  • the incubation time is in this case known to be between 15 and 20 minutes, in particular about 18 minutes.
  • the enzyme trypsin has a harmful influence on the cell vitality of epithelial cells, in particular keratinocytes, after just a few minutes.
  • the enzyme reaction is stopped after the end of the defined incubation period. The suspension is passed through a cell screen, centrifuged off, and the cells are resuspended in a specific nutrient medium.
  • tissue layer in particular the dermis layer, from which the epithelial layer has been removed, is optionally prepared.
  • the technical problem underlying the present invention is the provi- sion of an automatable method for separating and isolating a tissue layer, particularly an epithelial cell layer, from other tissue layers of a tissue cluster consisting at least of a first tissue layer, preferably an epithelial layer containing epithelial cells, and at least one further tissue layer, wherein preferably the epithelial layer is connected to the at least one further tissue layer via a basal membrane arranged between the epithelial layer and the further tissue layer.
  • the method is primarily intended to avoid technically complex handling steps which are difficult to automate, such as are known from the manual processing of pieces of tissue, and to allow a simple technical implementation in automated, robot-assisted systems.
  • Epidermis cells are intended to be separated and in particular isolated from a piece of biological tissue.
  • the piece of tissue preferably is a donor tissue, preferably human or animal skin tissue, preferably comprising at least one epidermis and at least one dermis layer.
  • a technical problem related thereto is the provision of a specific device for carrying out this method.
  • the technical problem is solved by providing a method for separating a first tissue layer from a multilayered biological tissue cluster, consisting of the first tissue layer and at least one further tissue layer connected, in particular tightly, to the first tissue layer, wherein the first tissue layer is mechanically more brittle compared to the further tissue layer and thus the further tissue layer is mechanically more ductile or tougher compared to the first tissue layer.
  • the method is characterized in that a brittle fracture is triggered in the first tissue layer by applying one or more sudden force pulses (chopping) to the tissue cluster, thus allowing the first tissue layer, which is split open as a result, to be detached from the further tissue layer.
  • the method according to the invention allows an agent detaching the tissue layers at their connecting points to penetrate through the brittle fracture of the integrated first tissue layer which would otherwise prevent the agent from advancing up to the tissue connecting points.
  • the method according to the invention also allows the first tissue layer, which is disintegrated as a result of the brittle fracture, to be able to be detached in ..fragments" from the at least one further tis- sue layer, so that simple subsequent handling steps allow the tissue layers to be separated.
  • the tissue fragments of the first tissue layer are present in suspension, while the further tissue layers, which are not encompassed by the brittle fracture, remain intact as substantially continuous tissue and can thus easily be separated from the suspension.
  • a method for the isolation of epithelial cells from a tissue cluster consisting of an epithelial cell layer containing epithelial cells and at least one further tissue layer connected to the epithelial cell layer via a basal membrane positioned therebetween, which method is characterized by at least the following method steps:
  • a) The integrity of the epithelial cell layer of the tissue cluster is destroyed by, preferably repeated, chopping movements of a blade, that is by applying one or more sudden force pulses to the tissue cluster via the blade, so that the epithelial cell layer is split open, particularly at the site of the acting force pulse, preferably substantially, up to the basal membrane, wherein preferably the at least one further tissue layer positioned therebelow remains com- pletely, or preferably substantially, intact;
  • tissue cluster having the split-open epithelial cell layer into contact with an agent dissolving the basal membrane of the tissue cluster, so that the epithelial cell layer is detached from the tissue cluster, a residual tissue, containing the at least one further tissue layer, of the tissue cluster remaining completely, preferably substantially, intact; c) separating the detached epithelial cell layer from the residual tissue, containing the at least one further tissue layer, of the tissue cluster.
  • the chopping that is to say the applying of a sudden force pulse to the tissue cluster, in step a) takes place recurrently.
  • the repetition frequency of the chopping is in step a) from 1 to 5 Hz, preferably from 3 to 5 Hz.
  • the person skilled in the art aware of the solution according to the invention can adapt other repetition rates, depending on the properties of the tissue cluster to be processed, in order if appropriate to improve the chopping result, without thereby departing from the idea of the invention.
  • the invention therefore makes provision to destroy the integrity of the epithelial cell layer by way of a sudden force pulse acting on the tissue cluster (chopping) before the tissue cluster is dissolved by a dissolving agent.
  • a sudden force pulse acting on the tissue cluster chopping
  • the at least one further tissue layer connected to the epithelial cell layer is not disintegrated or not substantially disintegrated, and therefore remains completely or at least substantially intact, is characteristic of this novel disintegration method. That is, following step a), the tissue cluster remains mechanically maintained as such; this facilitates automated, in particular robot-assisted, further processing in one piece.
  • the invention draws on the surprising finding that the mechanical properties of the epithelial cell layer differ from the mechanical properties of further tissue layers below and connected to the epithelial cell layer.
  • the epithelial cell layer is comparatively brittle compared to underlying further tissue layers connected thereto, and the further tissue layers clinging to an epithelial cell layer are comparatively ductile.
  • the sudden chopping pulse acting on the tissue causes a substantially deformationless brittle fracture in the epithelial cell layer, while comparatively ductile or tough tissue layers remain substantially intact.
  • the sudden chopping pulse cannot generate a high-deformation ductile fracture in a comparatively more ductile or tougher tissue.
  • the present invention allows the person skilled in the art to replace the splitting, which, as is known, in the past was achieved only by way of a drawing cut, of the tissue with a mechanical fracturing stress for separating cell layers, in particular the epithelial cell layer, in a tissue cluster.
  • the disintegration of the cell layer achieved thereby allows a tissue-dissolving agent to act directly on the basal membrane abutting the epithelial cell layer, as a result of which the basal membrane can be readily dissolved in order to dissolve the tissue cluster and in particular to detach or "dissolve" the epithelial cells from the at least one further tissue layer of the tissue cluster.
  • the advantageous effect of the suddenly acting force pulse (chopping) on the destruction of the integrity of the epithelial cell layer in the tissue cluster is in fact substantially independent of the orientation of the piece of tissue in relation to the chopping direction. It has been found that it is not, as would first have been expected, necessary for the cell layer to be disintegrated to have to be arranged on the surface of the tissue cluster that faces the direction of the force pulse in order to bring about, by way of the force pulse according to the invention, the disintegration of that layer. It has surprisingly been found that the at least one further tissue layer, which is connected to the epithelial cell layer in the tissue cluster, can also conduct said force pulse action onto the epithelial cell layer in order to disintegrate said layer.
  • the epithelial cell layer can in this way be ,,cut open" or incised in the tissue cluster without the specific orientation of the piece of tissue having to be defined or known in advance.
  • This is a further decisive advantage over the known measures of a drawing cut on the piece of tissue which must be oriented and spread out in a distinct manner.
  • the invention preferably provides for the tissue cluster to be chopped in step a) within the method in a manner distributed over substantially the entire surface of the tissue cluster in order to disintegrate, as far as possible, the entire epithelial cell layer, that is to say to split it open in substantial parts substantially up to the basal membrane.
  • this is achieved in that the tissue cluster rests in step a) on a carrier face which is moved, that is to say displaced, preferably step-by step, substantially perpendicularly to the chopping movement, that is to say perpendicularly to the direction of the force pulse exerted by the blade.
  • a cross table i.e.
  • the cross table has a first horizontal axis and a second horizontal axis lying perpendicularly thereto.
  • the invention preferably provides for the carrier face, and thus the piece of tissue resting thereon, to be moved, that is to say displaced, preferably step-by step in at least one axial direction, at least in the intervals between the recurring chopping movements, so that with each chopping movement the sudden force pulse of the blade is applied to a different surface portion of the tissue cluster resting on the carrier face.
  • the step-by-step movement is carried out firstly along at least one horizontal axis in the form of a regular pattern (scanning).
  • the movement is carried out in distribution patterns which are disordered in terms of at least one axial direction, in particular quasi-stochastic distribution patterns.
  • the advantage of the quasi-stochastic distribution of the force pulse action onto the surface of the tissue cluster consists in the fact that an on average balanced disintegration effect is achieved irrespective of the specific position of the tissue cluster on the carrier face.
  • the invention preferably provides for the agent dissolving the basal membrane in the tissue cluster that is used to be an enzyme or an enzyme mixture which is preferably selected from the enzyme dis- pase, dispase-like enzymes and mixtures thereof.
  • the tissue cluster is brought into contact with the agent dissolving the basal membrane in step b) by way of an incubation of the tissue cluster with this agent.
  • the incubation is carried out preferably over a period of from 3 to 5 hours, particularly preferably over a period of 4 hours.
  • the invention also provides a method which is supplemented by at least one further step d) in which the epithelial cell layer separated in step c) is brought into contact with a further agent dissolving the cells of the epithelial cell layer, so that singled-out epithelial cells detach or disintegrate from the separated epithelial cell layer.
  • the agent dissolving the cell cluster of the epithelial cell layer is an enzyme or enzyme mix which is preferably selected from the enzyme trypsin, trypsin-like enzymes and mixtures thereof.
  • the separated epithelial cell layer is brought into contact with the agent dissolving the cell cluster of the epithelial cell layer in step d) by way of an incubation of the epithelial cell layer with this agent.
  • the incubation is carried out over a period of preferably from about 3 to about 10 minutes, preferably from 4 to 6 minutes, particularly preferably of 5 minutes.
  • the invention also provides a method according to the invention which is extended by at least one further step, which is in a subsequent or alternative step e), in which the residual tissue having the at least one further tissue layer is brought into contact with an agent dissolving the tissue cluster, so that singled-out tissue cells of the at least one further tissue layer become detached or disintegrated from the residual tissue.
  • the agent dissolving the cell cluster of the at least one further tissue layer in step e) is an enzyme or enzyme mix, selected from the enzyme collagenase, collagenase-like enzymes and mixtures thereof.
  • the at least one further tissue layer is brought into contact with the agent dissolving the cell cluster of the tissue layer in step e) by way of an incubation of the at least one further tissue layer in the agent.
  • the incubation is carried out prefera- bly over a period of from 1 to 8 hours, particularly preferably over a period of from 3 to 5 hours.
  • the yield of the singled-out tissue cells detached from the cell cluster of the at least one further tissue layer is dependent on the incubation period.
  • the invention thus provides an overall method which allows a sim- pie, rapid and reliable isolation from a tissue cluster, preferably in the form of a donor tissue, which contains at least two tissue layers and has an epithelial cell layer and a further tissue layer connected via a basal membrane, both of the epithelial cells and also preferably, in addition, of the tissue cells of the further tissue layer from the tissue cluster, in an automatable, in particular robot-assisted manner.
  • the method of this type is reproducible, satisfies high- throughput requirements and can fulfil the demands placed on GMP conformity.
  • the tissue cluster is a donor tissue of the human or animal body; particularly preferably, the tissue cluster is skin tissue, in particular a skin tissue biopsate.
  • the epithelial cell layer is preferably an epidermis layer and the epithelial cells to be isolated are preferably keratinocytes which can be isolated as such from the donor tissue, in particular skin tissue.
  • the at least one further tissue layer of the tissue cluster is preferably a dermis layer and the tissue cells which can be isolated from the at least one further tissue layer are preferably fibroblasts.
  • a further subject matter of the invention is a device, which is suitable for carrying out the method according to the invention, in particular for carrying out step a) according to the invention, for destroying the integrity of an epithelial cell layer in a piece of tissue of a tissue clus- ter, consisting of an epithelial cell layer containing the epithelial cells and at least one further tissue layer connected to the epithelial cell layer via a basal membrane positioned therebetween, by applying a sudden force pulse (chopping) to the tissue cluster via a blade.
  • the device comprises at least the following components:
  • a carrier face (10) which is arranged via a cross table or scanning stage mount on a base (40) which is suitable for receiving the piece of tissue and has a first horizontal axis of movement (42) and a second horizontal axis of movement (44) arranged perpendicularly thereto;
  • a drive element (35) which is force-connected to the blade lever (30) in order to allow a sudden lowering of the at least one blade (20) onto a piece of tissue received on the carrier face (10).
  • the blades used are scalpel knives
  • these are exchangeable scalpel blades
  • the device is preferably characterized in that the at least one blade can be lowered substantially perpendicularly onto the carrier face. This is brought about by way of corresponding arrangements of the blade lever which is pivotably mounted on the base The pivot axis of the blade lever lies preferably above the plane of the carrier face.
  • the drive element provided for driving the blade lever is a force drive which is preferably selected from pneumatic drives, hydraulic drives and piezoelectric drives and electromagnetic drives.
  • the subject matter of the invention also includes drive designs similar thereto.
  • the electromagnetic drive is particularly preferred
  • a preferred feature of the drive element is the capacity to apply a sudden force pulse to a piece of tissue via the blade.
  • the pivotable mounting of the blade lever is dispensed with in order to generate a direct connection between the blade and drive element.
  • the blade lever is dispensed with and the drive element is con- nected directly to the blade.
  • a particularly preferred embodiment of the device is characterized in that an end stop, which delimits toward the carrier face the stroke movement which can be exerted as a result of the lowering of the blade onto the carrier face, is provided at least at a site of the active mechanism connecting the blade to the drive element via the blade lever.
  • the blade it is preferably possible for the blade not to be lowered or not to be lowered completely onto the carrier face during the stroke movement, that is to say during the chopping according to the invention.
  • the surface of said carrier face is advanta- geously spared and wear to the carrier face and/or blade is minimised.
  • the end stop which is preferably provided allows complete severing of the piece of tissue resting on the carrier face, on account of a knife-cutting action caused by the blade, to be avoided or prevented.
  • This invention foregrounds not the knife- cutting action, generated by the descending blade, against the carrier face, but rather preferably the sudden force pulse which is generated by the blade in the tissue and leads to ..shattering" of the comparatively brittle epithelial tissue in the tissue cluster of the piece of tissue.
  • the device is therefore de- signed in such a way as to rule out a knife-cutting action of the blade.
  • the device is therefore also configured in such a way as to prevent what is known as a drawing cut on the piece of tissue.
  • the person skilled in the art is familiar with appropriate geometrical configurations, in particular the arrangement of the pivot axis of the blade lever in relation to the carrier face and the angle of attack of the blade descending onto the carrier face in order to avoid corresponding cutting movements run- ning substantially parallel to the surface of the piece of tissue.
  • the subject matter of the invention is also a device also having a control unit, in particular suitable for controlling the method sequence in step a) of the method according to the invention, which device is connected, preferably operatively connected and/or con- trol-connected, to the drive element (35) and effectors of the horizontal cross table axes (42, 44) and is specifically embodied to coordinate the displacement of the carrier face (10) along the axes (42, 44) with the stroke movement, performed by the blade (20) via the drive unit (35), onto the carrier face, in particular in such a way that, after preferably each performed stroke movement of the blade, the carrier face is displaceable, preferably step-by step, in at least one of the two horizontal axes (42, 44).
  • the control unit is preferably selected as a function of the drive unit used. If, for example, a pneumatic drive unit is used, the control unit is selected from pneumatic control elements, if appropriate in conjunction with program-controlled valves.
  • the control unit can also be embodied by a purely mechanical operative connection, for example a mechanical latching or step sequence control, which is clocked via the stroke movement of the blade. Particularly preferred is a control implemented via a computer, preferably in conjunction with a computer program which is suitable for this purpose and is implemented on the control computer.
  • the subject matter of the invention is therefore also a computer program product for controlling the coordination of the movement of the blade with the movement of the carrier face.
  • the subject matter of the invention is therefore also an arrangement for the automatic, in particular robot-assisted isolation of epithelial cells from a tissue cluster consisting of or containing an epithelial cell layer containing the epithelial cells and at least one further tissue layer connected to the epithelial cell layer via a basal membrane positioned therebetween, the device containing at least the following components:
  • the means for destroying the integrity of the epithelial cell layer of the tissue cluster is the device according to the invention as described in the present document.
  • the arrangement also contains:
  • Figure 1 shows a preferred embodiment of the device according to the invention with a carrier face (10) which is mounted on the base (40) via the cross table with the horizontal axes (42, 44) and can be lowered onto at least one of the blades (20), preferably perpendicularly and preferably without movement components extending parallel to the carrier surface (10).
  • the at least one blade (20) is fixed in a force-transmitting manner via a blade block (25) to the end of a blade lever (30), which is pivotably mounted on the base (40), that points toward the carrier face.
  • the blade lever (30) acts as a lever for reversing the force effect generated by the drive element (35) which is illustrated in the present document in the form of a lifting magnet or a pneumatic piston.
  • the at least one blade (20) is lowered onto the carrier face (10).
  • the carrier face (10) is suitable for receiving the piece of tissue to be processed. If the piece of tissue is located on the carrier face (10), the at least one blade (20) descends, during the stroke movement of the unit formed from the elements (35, 30 and 20), onto the piece of tissue and exerts thereon a sudden force pulse without cutting up the piece of tissue by way of a knife-cutting action.
  • a handling element (46) which allows the automatic delivery of the carrier face (10) from and feeding of said carrier face into the device by way of a rotor-assisted system, is optionally associated with the carrier face (10).
  • Figure 2 depicts a different view of the device of Figure 1 with a blade clamped into the blade lever on the blade block. A piece of tissue rests on the square carrier plate.
  • pieces of skin tissue are removed, preferably by manual operation, from the human or animal body, preferably from the living human body. Preferably, these are prepuces.
  • the pieces of tissue are located at the beginning of the process preferably in a transport cup in approx. 20 ml of transport medium (preferably DMEM + 1 % gentamicin).
  • transport medium preferably DMEM + 1 % gentamicin.
  • the biopsate is first grasped by a gripper which takes the biopsate from the transport cup and deposits it onto a processing platform.
  • the transport cup is tilted, preferably by approx. 40° from the perpendicular axis. Gravity causes the biopsate to descend into the depression which has in this way formed in the transport cup, and thus into a reproducibly defined position.
  • the biopsate can in this way be grasped by a correspondingly designed gripper.
  • the biopsate is deposited, using the gripper, on a processing platform and prepared for the separation of the tissue layers.
  • the mechanical properties of the fatty tissue in relation to the dermis and epidermis tissue, in particular the modulus of elasticity, are drawn on for the purposes of fat reduction, i.e. for separating fatty tissue clinging to the biopsate.
  • the biopsate is firstly automatically transferred, preferably by means of reduced-pressure suction, to a cylindrical sleeve which is positioned above the bottom of a disposable vessel (Petri dish) in such a way that a gap of less than 1 mm is produced between the bottom of the vessel and the cylinder wall.
  • a piston or plunger running in the cylinder sleeve is used to press the biopsate onto the bottom.
  • the narrow gap between the cylinder and bottom can be penetrated only by the fatty tissue which is pressed to the side, in this way pushed outward through the gap and spatially separated from the remaining tissue (epidermis and dermis layer).
  • the issuing fatty tissue is removed by cutting-off.
  • a cutting ring which cuts off the fatty tissue protruding under the cylinder sleeve, is lowered at the outer side of the cylinder sleeve.
  • the cutting ring rotates about the axis of the cylinder sleeve in order to improve the cutting action by way of the drawing cut.
  • the cutting ring is mechanically coupled to the plunger extending in the interior of the cylinder sleeve, so that the outwardly protruding fatty tissue is cut in one operation immediately after the biopsate has been pressed down onto the bottom of the vessel.
  • the squeezing and cutting are implemented within a single stroke movement via a specifically designed entrainer sleeve which is coupled to the plunger and the cutting ring.
  • Spacers or webs, which determine the width of the gap, are provided for setting the cylinder sleeve apart from the bottom of the vessel.
  • the gap has a width of approx. 0.8 mm.
  • the fat separator has an intake line for applying a reduced pressure, via which the biopsate can be secured in the cylinder sleeve.
  • the biopsate can be drawn in from a defined position, be processed and be returned to a defined location by positioning the fat separator.
  • the treatment according to the invention provides, in addition to the reduction of the fatty tissue content of the biopsate, a flattening which is beneficial for further processing and planar spreading of the biopsate. This has a surprisingly positive influence on the efficiency of the further processing, in particular the separation of the tissue layers (dermis layer, epidermis layer).
  • the two skin layers, the epidermis and dermis are separated from each other. This takes place enzy- matically via the enzyme dispase or an enzyme which has a similar effect and causes a dissolution of the basal membrane arranged between the epidermis and dermis layers.
  • the biopsate is prepared in such a way that the enzyme can penetrate the tissue up to the basal membrane.
  • a tissue chopper which allows the biopsate, which has spread out in a planar manner, to be cut to a defined cutting depth, is preferably used for this purpose.
  • the surface of the biopsate consisting of the epidermis is severed only down to the dermis tissue positioned therebelow, that is to say the dermis tissue positioned therebelow remains preferably uninfluenced by the mechanical cutting.
  • the chopping movement of the blade causes the epidermis to be mechanically acted on from above at a high pulse, so that the epidermis virtually ..shatters" while the lower dermis layer remains substantially intact.
  • the size of the epidermis pieces is determined in particular by the ratio of the chopping frequency of the knife head and the speed of movement of the knife head above the biopsate and also by the duration of the chopping process.
  • the cutting or chopping action of the knife which chops recurrently, preferably at a frequency of from approx. 2 to 5 Hz, is distributed onto the entire piece of tissue, preferably by displacing the piece of tissue on a cross table which is operated while being coupled to the chopping knife.
  • the cutting guidance is thus distributed regularly, but preferably quasi-stochastically on the entire surface of the tissue, thus producing a preferably narrow distribution of the sizes of the cut epidermis pieces.
  • incubation is performed in an enzyme solution, i.e. Dispase (4 units/ml, 20 ml), at 37 0 C for a period of 4 hours.
  • the enzyme can easily advance to the basal membrane through the cut-into epidermis layer.
  • the cut-into epidermis becomes detached in pieces from the substantially continuously intact dermis positioned therebelow.
  • the epidermis pieces preferably float to the surface.
  • the epidermis pieces suspended in dispase are in the next step separated from the dermis which is obtained as a continuous piece of tissue.
  • the volume of the dispase solution is first doubled or tripled (to 40 to 60 ml) by topping-up with buffer solution and the dermis is automatically removed from the suspension of the epidermis pieces by means of a tissue gripper and deposited into a separate vessel.
  • the epidermis tissue and dermis tissue are now present in separate vessels.
  • the epidermis pieces are separated with the aid of a tissue filter, which is attached to the underside of an automated pipette head, in that the suspension is suction-extracted through the pipette head via the tissue filter, wherein the epidermis pieces cling to the tissue filter.
  • the epidermis pieces secured to the tissue filter are then transferred, by means of buffer (PBS) by backflushing the tissue filter via the pipette head, to a further vessel and mixed there with a further enzyme solution (trypsin/EDTA, 1 ml, 0.5 %).
  • PBS buffer
  • trypsin/EDTA 1 ml, 0.5 %
  • the enzyme reaction of the trypsin is stopped by adding 10 % FCS.
  • the step which is conventional in the manual method, of homogenising the pieces of tissue prior to the enzymatic treatment may be dispensed with.
  • the reduced mechanical loading thereby achieved of the cells and the shortening of the time of incubation in trypsin to approximately 5 min lead to an increased cell yield and vitality com- pared to the conventional manual process.
  • the suspension which is obtained is thoroughly mechanically mixed (vortex) in a subsequent step, if appropriate for approx. 20 sec.
  • the suspension is firstly separated via the tissue filter which has a pore size of 100 ⁇ m and allows the individual cells to pass.
  • a second individual cell filter which is connected downstream of the tissue filter in the direction of suction flow, is inserted on the automated pipette head. The suspension is drawn into the pipette head, as a result of which the tissue remnants which may still be contained in the suspension are retained on the tissue filter and the individual cells are secured to the individual cell filter connected downstream.
  • the tissue filter together with the tissue remnants clinging thereto, is separated from the automatic pipette head and if appropriate discarded.
  • the individual cells remaining in the individual cell filter in the pipette head are flushed out of the pipette head by backflushing the individual cell filter.
  • a pipette tip instead of the tissue filter, is placed onto the pipette head and the individual cells are brought into the pipette tip by back- flushing the individual cell filter.
  • the number of cells in the suspension is determined.
  • a part of the individual cell suspension is metered from the pipette tip into a cell counting chamber and the total number of cells in the pipette tip is concluded from the number of cells found in this partial volume. If appropriate, new medium is added by exchanging the suspending solution in the pipette head as a function of the number of cells found, so that a cell suspension made up of epidermis cells having a defined cell concentration is obtained.
  • the dermis layer is incubated in a collagenase solution (20 ml) at 37 0 C for a period of from 1 to 8 hours.
  • the cell yield is inter alia dependent on the incubation period. As the incubation period increases, the degree of dissolution of the dermis tissue rises.
  • the fibroblasts which become detached from the tissue during the enzyme treatment, are separated by similar filtration steps using a tissue filter and individual cell filter, optionally using the automated pipette head, as presented hereinbefore for the epidermis cells.
  • non-dissolved dermis tissue parts are removed, preferably by means of an auto- mated gripper.
  • the dermis cells are then optionally set to a specific cell concentration by means of the automated pipette head.
  • the automated method is designed so as to be able to be operated in a GMP-compliant manner.
  • the modularity of the individual automation steps allows high flexibility with respect to the tissue used and the desired application, including the possibility of carrying out individual processing steps manually.

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Abstract

La présente invention concerne le domaine technique de l'ingénierie tissulaire. Elle concerne, plus précisément, un procédé de séparation et d'isolement automatisés de cellules tissulaires d'une couche tissulaire d'un tissu biologique multicouches d'un organisme humain ou animal, en particulier de cellules épithéliales de biopsies cutanées, ainsi qu'un dispositif permettant une mise en œuvre automatisée dudit procédé.
EP10706522A 2009-05-15 2010-02-20 Séparation automatisée de couches tissulaires Withdrawn EP2430155A1 (fr)

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DE200910022349 DE102009022349B4 (de) 2009-05-15 2009-05-15 Automatisches Trennen von Gewebeschichten
PCT/EP2010/001080 WO2010130305A1 (fr) 2009-05-15 2010-02-20 Séparation automatisée de couches tissulaires

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EP2430155A1 true EP2430155A1 (fr) 2012-03-21

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DE102011002197B4 (de) * 2011-04-20 2019-03-07 Leica Biosystems Nussloch Gmbh Verfahren und Vorrichtung zum Ablösen und/oder Vereinzeln einer histologischen Probe
DE102011002195B4 (de) 2011-04-20 2020-07-02 Leica Biosystems Nussloch Gmbh Verfahren und Vorrichtung zum Ablösen und/oder Vereinzeln einer histologischen Probe
JP2013255447A (ja) * 2012-06-12 2013-12-26 Nippon Koden Corp 細胞単離装置
CN105241686B (zh) * 2015-08-10 2018-04-13 河南科技大学 小鲵科动物视网膜显微组织切片的制备方法
CN106895990A (zh) * 2017-04-24 2017-06-27 湖北省疾病预防控制中心 一种猪皮皮肤表皮和真皮层分离取材的方法
WO2021100047A1 (fr) * 2019-11-22 2021-05-27 Cutiss Ag Procédés et systèmes de production de greffons cutanés
WO2023181636A1 (fr) * 2022-03-23 2023-09-28 富士フイルム株式会社 Procédé d'isolement cellulaire et appareil d'isolement cellulaire

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DE19912798C1 (de) * 1999-03-10 2000-02-17 Andreas Jordan Verfahren zur Kultivierung von Krebszellen aus Humangewebe und Vorrichtung zur Aufbereitung von Gewebeproben
US8741635B2 (en) * 2004-05-12 2014-06-03 The Walter And Eliza Hall Institute Of Medical Research Method of cell isolation

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WO2010130305A1 (fr) 2010-11-18
DE102009022349B4 (de) 2011-02-03

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