Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/32—Surgical cutting instruments
  • A61B17/322—Skin grafting apparatus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
  • G01N1/08—Devices for withdrawing samples in the solid state, e.g. by cutting involving an extracting tool, e.g. core bit
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/32—Surgical cutting instruments
  • A61B17/3205—Excision instruments
  • A61B17/32053—Punch like cutting instruments, e.g. using a cylindrical or oval knife
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B2017/00969—Surgical instruments, devices or methods, e.g. tourniquets used for transplantation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/32—Surgical cutting instruments
  • A61B17/322—Skin grafting apparatus
  • A61B2017/3225—Skin grafting apparatus with processing of harvested tissue

Definitions

• the invention relates to a method for separating tissue clinging to donor tissue of the animal or human body, particularly fatty tissue, and also to a device for automatically carrying out this method.
• 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 donor tissue, which can be recovered in a separate method from the human or animal body in the form of donor tissue as what are known as biopsates, and in constituting new tissues therefrom.
• the isolated cells are propagated and subsequently applied in order to build up newly constituted three-dimensional tissue constructs, for example newly constituted skin equivalents.
• Newly constituted tissues of this type may then be used as test tools in research, in particular in the research of active compounds, or as transplants in medicine to replace lost organ functions.
• two-layered skin models are preferably used as test systems for active compounds, chemicals and cosmetics and provide an alternative to animal testing.
• fibroblasts and keratinocytes, which, as is known, may be isolated from a multilayered donor skin tissue, in particular prepuce biopsates.
• a particular application is the separation of fatty tissue, i.e. subcutaneous fat, clinging to skin tissue, thus providing a tissue cluster which contains substantially no fatty tissue and from which individual cells can be isolated.
• the fatty tissue clinging thereto is manually separated from the bi- opsate with the aid of a pair of forceps which grips in a defined manner and a scalpel.
• the fatty tissue is distinguished from other tissue layers of the donor tissue during the cutting purely under ongoing visual inspection.
• skin biopsates the fatty tissue is generally securely connected to the dermis and may be separated only by hand and in a time-consuming manner. The fact that skin biopsates tend to roll up further impedes the handling of donor tissue. The separation of fat reduces this tendency and the biopsate is easier to handle.
• the technical problem underlying the present invention is the provision of an automatable method for separating fatty tissue from a piece of biological tissue, the piece of tissue being preferably a donor tissue, particularly preferably human or animal skin tissue, which preferably has at least one epidermis and at least one dermis layer. Furthermore, the provision of a specific device for carrying out this method which above all avoids techni- cally complex handling steps which are difficult to implement, such as are known from the manual processing of donor tissue, and allows a simple technical implementation in automated, robot-assisted systems.
• a sleeve 10
• a basically tubular hollow shaft with an upper end
• a bottom element (40) basically a base plate, which covers the lower end (12) of the sleeve and is set apart from the sleeve (10), so that a peripheral, i.e. circumferential annular gap, is formed between the bottom element (40) and lower end (12) of the sleeve (50); and
• a plunger (30) which is arranged in the sleeve (10), preferably so as to be displaceably movable and closes off the sleeve (10) toward the upper end (14), wherein a space (60) is formed within the sleeve between the bottom element (40) covering the sleeve (10) at its lower end (12), the inner wall of the sleeve (16) and the plunger (30) a space (60) for receiving the donor tissue.
• the plunger (30) preferably can be lowered toward the lower end (12) of the sleeve in order to apply compressing pressure to a tissue received in the space (60).
• direct application of fluidic (pneumatic, hydraulic) pressure in the space (60) is provided instead of the displaceably movable plunger (30) in order to apply compressing pressure to a tissue received in the space (60).
• the device further comprises
• a cutting ring (20) which encircles the sleeve (10) at the outer side thereof and is arranged on the sleeve so as to be displaceably movable.
• the cutting ring (20) may comprise a revolving cutting wedge (25) pointing downward toward the bottom element (40).
• the cutting ring (20) can be lowered onto the bottom element (40).
• the cutting ring (20) may form with the lower end (12) of the sleeve a shearing active element.
• the cutting ring (20) may form, together with the bottom element (40) a knife-cutting active element.
• the cutting ring (20) is preferably arranged so as to be able to rotate co- axially about the axis of the sleeve for assisting the cutting action.
• the invention also concerns the use of the device according to the invention for separating fatty tissue clinging to a piece of biological tissue.
• the technical problem is also solved by the provision of a method for separating fatty tissue clinging to a piece of biological tissue, the piece of tissue being preferably a donor tissue, particularly preferably human or animal skin tissue, which preferably has at least one epidermis and at least one dermis layer.
• the method according to the invention comprises at least the following steps which are preferably carried out in the specified order, preferably in immediate succession:
• the piece of tissue is held, preferably by means of reduced pressure, that is to say by drawing-in, on or in the device according to the invention and then preferably brought onto the bottom element.
• the automatable drawing-in of the piece of tissue allows simpler handling options and makes it possible to carry out, if appropriate, a plurality of tis- sue separating steps in succession.
• the method is carried out using the device according to the invention which is specifically embodied for this purpose.
• the first region, to which pressure is applied is separated from the adjoining second region, to which no pressure is applied, by a sleeve or a basically hollow shaft which is arranged above the piece of tissue, is set apart from the bottom element and forms at its lower end with the bottom element a circumferential annular gap. Both regions are connected across the gap between the bottom element and sleeve, so that the piece of tissue to which pressure is applied inside the sleeve can at least partly issue or escape therefrom through the gap.
• pressure is applied by lowering a plunger, which is preferably guided so as to be able to move within the sleeve arranged on the piece of tissue, onto the piece of tissue resting on the bottom element.
• the invention also provides a method for the isolation of fatty, i.e. adipose tissue from a donor tissue of the human or animal body. This method preferably serves to recover stem cells from the isolated adipose tissue separated from the donor tissue.
• the method according to the invention is characterized by the steps:
• adult stem cells are isolated in a manner known per se from said separated fatty tissue.
• the invention also relates to the fatty tissue which can be obtained from donor tissue in this manner according to the invention as well as to the adult stem cells which can be obtained therefrom.
• the invention also concerns the use of the device according to the invention for recovering fatty tissue from donor tissue of the human or animal body, and preferably the subsequent recovery of stem cells, particularly adult stem cells from the fatty tissue.
• the invention also provides for a general arrangement for the automatic isolation of biological cells from a piece of biological tissue, which arrangement contains at least the device according to the invention apt for separating fatty tissue from the piece of tissue and is in particular characterized thereby.
• the arrangement according to the invention preferably also contains in addition:
• the invention draws on the finding that the mechanical properties of a piece of tissue, on the one hand, and the fatty tissue clinging or attached thereto, on the other hand, in particular the modulus of elasticity, are so different that applying pressure to the piece of tissue resting in a substantially planar manner allows the fatty tissue then to exclusively be made spatially separate and subsequently to be separated from the remainder tissue.
• the piece of tissue in particular a skin tissue, comprising particularly an epidermis layer and a dermis layer, has a comparatively non-resilient, tough structure.
• the fatty tissue attached thereto is comparatively resilient and yields to compressive loading, like a resilient elastically fluidic substance.
• Figure 1 shows a device according to the invention for separating fatty tissue from biological tissue in a lateral sectional view ( Figure 1a) and in a plan view ( Figure 1b).
• the embodiment according to Figure 1 comprises a preferably cylindrical sleeve (10) with an upper end (14) and a lower end (12).
• the lower end (12) of the sleeve (10) is adjoined by a bottom element (40) which at least completely covers the lower end (12) of the sleeve (10).
• the bottom element (40) is spaced apart from the sleeve (10) in such a way that a gap (50) is formed between the bottom element (40) and the lower end (12) of the sleeve (10).
• a preferably displaceably movable plunger (30) is arranged within the sleeve (1O) 1 .
• the plunger (30) closes off the sleeve (10) toward the upper end (14) of the sleeve so as to produce a seal.
• the space (60) communicates, i.e. is in fluid connection, with the outer side of the sleeve via the preferably circumferential peripheral gap (50).
• the device is configured in such a way that it is pos- sible to compensate for an application of pressure in the space (60) via the gap (50).
• the space (60) serves to receive the biological tissue which preferably rests on the bottom element (40) in a substantially planar manner.
• a preferred circular concentric arrangement is shown in Figure 1 , but can be replaced by a different cross-sectional shape, for example ellipsoidal, square or rectangular, without these shapes prejudicing alternative embodiments outside the idea of the invention.
• Such alternative embodiments form also the subject-matter of the invention.
• a device preferably further comprises an annu- lar cutting ring (20) which is arranged circumferential to the outer side of the sleeve (10), and is arranged on the sleeve (10) in such a way as to be displaceably movable thereon.
• the cutting ring (20) comprises at its underside, that is to say the side facing towards the bottom element (40) and the lower end (12) of the sleeve, a circumferential revolving cutting wedge (25).
• the cutting wedge is used for improved separating of the fatty tissue (110) issuing or escaping the sleeve through the gap (50) when pressure is applied to the biological tissue (100) located in the space (60) ( Figure 2b).
• the cutting ring (20) can be lowered onto the bottom element (40) ( Figure 2c).
• the annular cutting ring (20), proximate to the gap (50), forms with the lower end (12) of the sleeve a shearing active element or tool which serves to separate the issuing fatty tissue.
• the cutting ring (20) it is prefer- able for the cutting ring (20) to form, proximate to the gap (50), with the bottom element (40) a knife-cutting active element or tool which serves to cut off the issuing fatty tissue, the bottom element (40) forming the rest and the cutting ring (20) forming, preferably in conjunction with the cutting wedge (25), a knife-cutting tool.
• a revolving annular knife is formed.
• the cutting wedge (25) is structured, preferably toothed or serrated, at its cutting edge in order to improve the cutting action.
• the cutting edge is bevelled in order to cause an oblique cut when the cutting ring (20) is lowered in the tissue to be separated.
• the cutting ring (20) is arranged so as to be able to rotate co- axially about the axis of the sleeve, in particular for assisting the cutting action, and exerts, preferably during the cutting process, a rotational movement running preferably substantially perpendicularly to the axis of the sleeve, thus causing a drawing cut on the issuing fatty tissue.
• the plunger (30) which is arranged inside the sleeve, preferably so as to be displaceably movable, can be lowered toward the bottom element (40), i.e. toward the lower end (12) of the sleeve (10), for applying pressure to the tissue received in the space (60).
• the pressure on the tissue is increased as a result of the lowering of the plunger (30).
• a surface pressure acting perpendicularly on the tissue preferably resting on the bottom ele- ment (40) in a substantially planar manner is preferred.
• a direct fluidic, preferably pneumatic and/or hydraulic, application of pressure into the space (60) is provided, preferably instead of a displaceably movable plunger (30). Fluidic pressure is applied preferably by way of an inflow into the space (60) which is preferably fluidically connected to a pressure pump or pressure receiver.
• the gap (50) which is provided in accordance with the invention has a gap width resulting from the setting-apart of the sleeve (10) and bottom plate (40).
• the gap width is 1 mm or less, preferably less than 1 mm and more than 0.4 mm.
• the gap (50) there to be provided in the region of the gap (50) at least one web (55) which connects the sleeve (10) to the bottom plate (40) with a spacing in order to define the gap width substantially as a spacer element.
• a plurality of webs (55) are provided, preferably set uniformly apart, along the gap (50), encircling the lower end (12) of the sleeve.
• the peripheral gap (50) is provided as apertures in the sleeve (10), at the lower end (12) thereof.
• the device according to the invention further comprises at least one means for generating a reduced pressure, in particular a suction-extraction line which is preferably fluidically connected to a vacuum receiver or vacuum pump.
• a suction-extraction line which is preferably fluidically connected to a vacuum receiver or vacuum pump.
• Figure 2 illustrates one mode of operation of the device according to the invention in the method according to the invention for separating fatty tissue hanging from a piece of biological tissue.
• the piece of biological tis- sue (100) rests, together with the fatty tissue (110) adjacent thereto, in a substantially planar manner on the bottom plate (40) ( Figure 2a).
• sleeve (10) Spaced apart from the bottom plate (40), sleeve (10) is arranged above the piece of tissue (100).
• the sleeve has an inner plunger (30), which is preferably arranged so as to be displaceably movable, and preferably one cutting ring (20) which is arranged on the outer circumference of the sleeve (10) and preferably comprising an annular cutting wedge (25).
• the sleeve (10) and bottom plate (40) form a gap (50) which separates a first region (120) located within the sleeve from an adjacent second region (130) located outside the interior of the slee
• Figure 2b illustrates how, according to the invention, surface pressure is applied to the piece of tissue (100) by pressing-on the plunger (30) in the region (120).
• the piece of tissue (100) is pressed onto the bottom plate (40), wherein at the same time the fatty tissue (110) clinging to the piece of tissue (100) is pressed out through the gap (50) into the region (130) to which no pressure is applied.
• the piece of tissue with the fatty tissue clinging thereto to be arranged on the bottom plate in such a way that the fatty tissue or layer of fatty tissue points toward the bottom plate.
• the fatty tissue which has issued in the region of the gap (50) is separated from the piece of tissue by lowering the cutting ring (20).
• the cutting ring Preferably, for assisting the cutting process, provision is made for the cutting ring to move, preferably within the cutting process, preferably so as to additionally rotate about the longitudinal axis of the sleeve (10) in order to improve or to facilitate, by way of a drawing cut, the separation or blanking of the issuing fatty tissue ( Figure 2c).
• pressure is applied by direct application of fluidic pressure, selected from pneumatic and hydraulic pressure, onto the piece of tissue.
• fluidic pressure selected from pneumatic and hydraulic pressure
• the issuing fatty tissue is separated by a revolving annular knife.
• pieces of skin tissue are removed, preferably by manual operation, from the human or animal body, preferably from the living human body. Preferred 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 biop- sate 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 then deposited, using the gripper, on a processing platform and prepared for the separation of the tissue layers.
• the mechanical properties of the adipose 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 automatically transferred, by means of reduced pressure applied in the device, to a cylindrical sleeve which is posi- tioned 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 tis- sue 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 and planar spreading of the biopsate which is beneficial for further processing. 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 enzymatically 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. 1 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.
• the epidermis pieces suspended in dispase are, in a 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 at- tached 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 (10 ml or 20 ml of 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 or 2 ml, 0.5 %, 1 :10) and thoroughly mechanically mixed, i.e. vortexed.
• 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.
• 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 compared 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 backflushing 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.
• 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 automated 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.

Applications Claiming Priority (2)

Publications (1)

Family

ID=42199920

Family Applications (1)

Country Status (3)

Families Citing this family (3)

Family Cites Families (11)

• 2009
  • 2009-05-15 DE DE200910022346 patent/DE102009022346B4/de not_active Expired - Fee Related
• 2010
  • 2010-02-20 EP EP10706152A patent/EP2429425A1/de not_active Withdrawn
  • 2010-02-20 WO PCT/EP2010/001079 patent/WO2010130304A1/en active Application Filing

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events