WO2010130305A1

WO2010130305A1 - Automated separation of tissue layers

Info

Publication number: WO2010130305A1
Application number: PCT/EP2010/001080
Authority: WO
Inventors: Frank Pretzsch; Ulrike Koropp; Andrea Heymer
Original assignee: Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
Priority date: 2009-05-15
Filing date: 2010-02-20
Publication date: 2010-11-18
Anticipated expiration: 2011-11-15
Also published as: WO2010130305A8; EP2430155A1; DE102009022349B4; DE102009022349A1

Abstract

The invention relates to the technical field of tissue engineering. It concerns 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, and also a device for automatically carrying out this method.

Description

Automated separation of tissue layers

Description

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. For example, two-layered skin models are preferably used as test systems for active ingredients, chemicals and cosmetics and provide an alternative to animal testing. It is expected that the high demand for skin test systems made up of human primary cells and the requirements placed on the reproducibility thereof can ultimately be ensured only by automation of the production process. The construction of skin test constructs requires at least two different primary cell types: fibroblasts and keratinocytes which, as is known, are isolated from a multilayered skin tissue, and in particular from prepuce biopsates.

Particularly in relation to regenerative medicine, there is the need to automate in a GMP-compliant manner biological laboratory proc- esses under clean room conditions. A higher yield, higher process safety and also standardisable process optimisation and process control are to be achieved in this way.

One difficulty exists above all in the conversion of the large number of different previously known manual steps and operational se- quences of tissue preparation and cell isolation into expediently automated handling operations. A particular challenge presented to the automation of sequences of this type is in this case the separation of various tissue layers from multilayered tissue clusters, in particular the separation of epithelial layers from tissue clusters such as skin biopsates.

In relation to the isolation of tissue layers of various cell types from donor tissue, in particular skin tissue, manual methods are known in which, for separating the epidermis and dermis of a skin biopsate immediately after the separation of any fatty tissue attached thereto using a scalpel, small pieces are cut to approximately 2 x 2 mm² and transferred to an enzyme solution. The purpose of the enzyme solution is to dissolve the connection between the epidermis and dermis, particularly the basal membrane arranged therebetween. Complete cutting-up of the biopsate is required in order to offer the enzyme a surface for acting on the basal membrane. This manual method is not automatable. The known method assumes that it is possible to cut up the biopsate only by way of a drawing cut on the epidermis side. Furthermore an orientation, required for a defined cut or incision, of the biopsate in a reproducible manner cannot be implemented on account of the different biopsate sizes and composition. A significant problem results above all from the fact that the cut-up or incised biopsate tissue then has to be separated manually into the two tissue layers. In the known manual methods, this is carried out on the individual biopsate pieces by means of a pair of forceps and under visual inspection, the pair of forceps being used to detach the epidermis layer from the somewhat thicker dermis layer. The fact that skin biopsates tend to roll up additionally impedes handling. There is at present no technology suitable for evaluating the qualitative visual difference between the two tissue layers for an automated, robot-assisted detachment of the tissue layers from one another.

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. A drawback of this is that the enzyme trypsin has a harmful influence on the cell vitality of epithelial cells, in particular keratinocytes, after just a few minutes. In the known method 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.

In addition, the further tissue layer, in particular the dermis layer, from which the epithelial layer has been removed, is optionally prepared.

Previous attempted approaches for automation in the field of cell culture technology and tissue engineering have been restricted mainly to the cultivation of individual cells. Automated robot systems are known that can handle cell culture flasks or bioreactors, in particular in the format of standardised multiwell plates. All the steps required for the cell culture (incubation, exchange of media, passaging and harvesting processes) are automated and, if appropriate, also carried out under GMP-compliant conditions. To date, automatable methods or means have been provided neither for the cell extraction of the cells to be cultivated, nor for the further processing of the cultivated cells.

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. In particular, 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. Particularly, 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.

As a preferred embodiment there is provided 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;

b) bringing the 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.

Preferably, 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. The fact that in this case 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. In particular, 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. Without wishing to be bound to the theory, 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 inventors have further surprisingly found that 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. Advantageously, 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. In a preferred variant of the invention 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. This is carried out preferably as a result of the use of a cross table, i.e. scanning stage, on which the carrier face may be arranged. 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. In a first variant the step-by-step movement is carried out firstly along at least one horizontal axis in the form of a regular pattern (scanning). In an alternative variant 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. Preferably, 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.

Preferably, 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. Preferably, 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.

Preferably, 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. Preferably, 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.

In a preferred embodiment 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. Accordingly, 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. Likewise, 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 blade lever (30) which is pivotably mounted on the base

(40) and comprises at least one blade block (25) which is ar- ranged at the end facing the carrier face (10) and receives at least one blade (20) which can be lowered by pivoting the blade lever

(30) onto the carrier face (10);

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).

Provision is preferably made for a plurality of blades to be arranged in the blade block In a preferred embodiment the blades used are scalpel knives Preferably, 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 In a preferred alterna- tive embodiment the pivotable mounting of the blade lever is dispensed with in order to generate a direct connection between the blade and drive element. In a further alternative preferred embodiment 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. As a result, 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. As a result, the surface of said carrier face is advanta- geously spared and wear to the carrier face and/or blade is minimised. At the same time, 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. In a preferred embodiment the device is therefore de- signed in such a way as to rule out a knife-cutting action of the blade. In a preferred embodiment of the device, 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:

means for destroying the integrity of the epithelial cell layer of the tissue cluster by applying a sudden force pulse

(chopping) to the tissue cluster via a blade, so that the epithelial cell layer is split open substantially up to the basal membrane;

means for bringing the tissue cluster with the split-open epithelial cell layer into contact with an agent dissolving the basal membrane, so that the epithelial cell layer is detached from the tissue cluster; and

- means for separating the detached epithelial cell layer from the residual tissue, containing the at least one further tissue layer, of the tissue cluster.

In a preferred embodiment 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. Preferably, the arrangement also contains:

means for separating fatty tissue from the donor tissue;

means for resuspending the singled-out cells and for separating the cells from tissue remnants which may be present.

The invention will be described in greater detail by way of the following figures and also the example, although these are not to be understood as entailing any limitation.

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. By extending the lifting piston from the drive element (35), 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.

Example: Fully automated isolation of fibroblasts and keratino- cvtes from epidermis and dermis layers of skin biopsates

In an optionally preceding separate method sequence which is carried out independently of the method according to the invention, pieces of skin tissue (biopsates) 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). Within the method according to the invention the biopsate is first grasped by a gripper which takes the biopsate from the transport cup and deposits it onto a processing platform. In order to bring the biopsate for automated gripping into a defined position, 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. In this case, 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. For this purpose, 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.

For automated receiving and handling of the biopsate, the fat separator has an intake line for applying a reduced pressure, via which the biopsate can be secured in the cylinder sleeve. As a result, the biopsate can be drawn in from a defined position, be processed and be returned to a defined location by positioning the fat separator. It has been found that 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).

For the separate isolation of the two types of cell, keratinocytes and fibroblasts, from the skin biopsate, 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. For this purpose, 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. In this case, 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. On account of the comparatively higher brittleness of the top epidermis layer in relation to the tougher dermis layer positioned therebelow, 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.

Then, incubation is performed in an enzyme solution, i.e. Dispase (4 units/ml, 20 ml), at 37 ⁰C for a period of 4 hours. The enzyme can easily advance to the basal membrane through the cut-into epidermis layer. As a result of the enzymatic destruction of the basal membrane, 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. For this purpose, 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.

For preparing the epidermis layer to form singled-out keratinocytes, 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 increasing of the volume of the solution as a result of the topping-up with buffer solution immediately before the filtration improves the epidermis yield considerably.

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 %). The trypsin causes the individual cells to become detached from the tissue cluster of the epidermis pieces. After incubation (for approximately 5 minutes) an individual cell suspension made up of epidermis cells is present.

At the end of the incubation time the enzyme reaction of the trypsin is stopped by adding 10 % FCS. As small epidermis pieces can already be present as a result of the optional chopping process, 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.

In order to assist the cell isolation, the suspension which is obtained is thoroughly mechanically mixed (vortex) in a subsequent step, if appropriate for approx. 20 sec. For separating any tissue remnants, the suspension is firstly separated via the tissue filter which has a pore size of 100 μm and allows the individual cells to pass. For this purpose, 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. Subsequently, 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. For this purpose, 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. Optionally, the number of cells in the suspension is determined. For this purpose, 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.

For preparing the dermis layer to form fibroblasts, the dermis layer is incubated in a collagenase solution (20 ml) at 37 ⁰C 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. Alternatively, 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.

Claims

1. 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 to the first tissue layer, the first tissue layer being mechanically more brittle compared to the further tissue layer and the further tissue layer being mechanically tougher compared to the first tissue layer, 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 de- tached from the further tissue layer.

2. Method according to claim 1 for the isolation of epithelial cells from a tissue cluster, 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 therebe- tween, containing the steps:

a) destroying the integrity of the epithelial cell layer of the tissue cluster by applying one or more sudden force pulses (chopping) to the tissue cluster via a blade, so that the epithelial cell layer is split open substantially up to the basal membrane; b) bringing the tissue cluster having the split-open epithelial cell layer into contact with an agent dissolving the basal membrane, so that the epithelial cell layer is detached from the tissue cluster; and

c) separating the detached epithelial cell layer from the residual tissue, containing the at least one further tissue layer, of the tissue cluster.

3. Method according to claim 1 or 2, wherein the tissue cluster is chopped recurrently.

4. Method according to claim 3, wherein the repetition frequency of the chopping is from 2 to 5 Hz.

5. Method according to one of the preceding claims, wherein the tissue cluster is chopped in a manner distributed over the surface of the tissue cluster.

6. Method according to claim 5, wherein the tissue cluster rests on a carrier face and the carrier face is moved substantially perpendicularly to the direction of the force pulse exerted by the blade.

7. Method according to one of claims 2 to 6, wherein the agent dissolving the basal membrane is in step b) an enzyme or an en- zyme mixture, selected from dispase, dispase-like enzymes and mixtures thereof.

8. Method according to one of claims 2 to 7, wherein the bringing of the tissue cluster into contact with the agent dissolving the basal membrane in step b) is an incubation of the tissue cluster in the agent over a period of from 3 to 5 hours.

9. Method according to one of claims 2 to 8, also having the step:

d) bringing the separated epithelial cell layer into contact with an agent dissolving the cell cluster of the epithelial cell layer, so that singled-out epithelial cells become detached from the epithelial cell layer.

10. Method according to claim 9, wherein the agent dissolving the cell cluster of the epithelial cell layer is in step d) an enzyme or enzyme mix selected from trypsin, trypsin-like enzymes and mixtures thereof.

11. Method according to claim 9 or 10, wherein the bringing of the epithelial cell layer into contact with the agent dissolving the cell cluster is in step d) an incubation of the epithelial cell layer in the agent over a period of from 4 to 6 min.

12. Method according to one of claims 2 to 11 , also having the step:

e) bringing the at least one further tissue layer of the residual tissue into contact with an agent dissolving the cell cluster, so that singled-out tissue cells of the at least one further tissue layer become detached from the residual tissue.

13. Method according to claim 12, wherein the agent dissolving the cell cluster of the at least one further tissue layer is in step e) an enzyme or enzyme mix, selected from collagenase, collagenase-like enzymes and mixtures thereof.

14. Method according to claim 12 or 13, wherein the bringing of the further tissue layer into contact with the agent dissolving the cell cluster is in step e) an incubation of the at least one further tissue layer in the agent over a period of from 1 to 8 hours.

15. Method according to one of the preceding claims, wherein the tissue cluster is a donor tissue from the human or animal body.

16. Method according to one of the preceding claims, wherein the tissue cluster is skin tissue.

17. Method according to one of the preceding claims, wherein the first tissue layer is an epidermis layer and the epithelial cells to be isolated are keratinocytes.

18. Method according to one of the preceding claims, wherein the further tissue layer is a dermis layer.

19. Method according to one of the preceding claims, wherein the cells of the at least one further tissue layer are fibroblasts.

20. Method according to one of the preceding claims, characterized in that in step a) a knife-cutting action of the blade is avoided.

21. Device for destroying the integrity of a first cell layer in a piece of tissue of a tissue cluster by applying a sudden force pulse (chopping) to the tissue cluster, comprising: - a carrier face (10), which is cross table-mounted on a base (40), for receiving the piece of tissue with a first horizontal axis of movement (42) and a second horizontal axis of movement (44) perpendicular thereto;

- a blade lever (30) which is pivotably mounted on the base (40) and has at least one blade (20) which is received in a blade block (25) arranged at the end facing the carrier face (10) and can be lowered by pivoting the blade lever (30) onto the carrier face (10);

- a drive element (35), which is force-connected to the blade lever (30), for suddenly lowering the at least one blade (20) onto a piece of tissue received on the carrier face (10).

22. Device according to claim 21 , characterized in that the drive element (35) is a force drive, selected from a pneumatic drive, a hydraulic drive, a piezoelectric drive and an electromagnetic drive.

23. Device according to claim 21 or 22, characterized in that a plurality of blades (20) are arranged in the blade block (25).

24. Device according to one of claims 21 to 23, character- ized in that the blade (20) can be lowered substantially perpendicularly onto the carrier face (10).

25. Device according to one of claims 21 to 24, 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 (20) onto the carrier face (10), is provided at least at a site of the active mechanism connecting the blade (20) to the drive element (35) via the blade lever (30).

26. Device according to one of claims 21 to 25, also comprising:

- a control unit which is connected to the drive element (35) and effectors of the horizontal cross table axes (42, 44), for controlling the displacement of the carrier face (10) along the axes (42, 44) in coordination with a stroke movement, performed by the blade (20) via the drive unit (35), onto the carrier face (10).

27. Device according to claim 26, characterized in that the control unit is specifically configured in such a way that, after each performed stroke movement of the blade (20), the carrier face (10) is displaceable step-by step in at least one of the horizontal axes (42, 44).

28. Arrangement for the automatic isolation of epithelial cells from a tissue cluster containing the components:

- means for destroying the integrity of the epithelial cell layer of the tissue cluster by applying a sudden force pulse (chopping) to the tissue cluster via a blade;

- means for bringing the tissue cluster with the split-open epithelial cell layer into contact with an agent dissolving the basal membrane; and - means for separating the detached epithelial cell layer from the residual tissue, containing the at least one further tissue layer, of the tissue cluster.

29. Arrangement according to claim 28, wherein the means for destroying the integrity of the epithelial cell layer of the tissue cluster is the device according to one of claims 21 to 27.

30. Arrangement according to claim 28 or 29, also containing:

means for separating fatty tissue from the donor tissue;

means for resuspending the singled-out cells and for separat- ing the cells from tissue remnants which may be present.