阅读说明：本技术 封闭组织解离和低温贮藏 (Closed tissue dissociation and cryopreservation ) 是由 G·J·莫里斯 S·兰布 于 2020-02-28 设计创作，主要内容包括：公开一种用于在封闭的柔性组织样本袋(10)中使组织样本解离为各个细胞或细胞团块的装置(100、200)；装置包括两个或更多个弹性脚(134/136、234/236),其顺序地踏压组织样本袋接收区域(148、248)。还公开一种用于将热能传递到区域(148、248)或从区域(148、248)传递热能的热传递板(150、250),板具有与区域(148、248)相邻的一个板表面(151、251)和背离区域(148、248)的暴露于外部热影响的相反表面(152、252)。另外公开一种组织样本接收袋(10),该组织样本接收袋包括一个或多个柔性塑料腔(12),其由两层塑料形成,该两层塑料围绕它们的边缘密封以形成大体上直线的周边,其中一个或多个腔(12)在周边内,且在周边的一侧处形成一个或多个可密封的通路端口(16)。袋的一部分留下不密封,以提供组织样本接收开口。(An apparatus (100, 200) for dissociating a tissue sample into individual cells or cell aggregates in a closed flexible tissue sample bag (10) is disclosed; the device includes two or more resilient feet (134/136, 234/236) that sequentially step against tissue specimen bag receiving regions (148, 248). A heat transfer plate (150, 250) for transferring thermal energy to or from a zone (148, 248) is also disclosed, the plate having one plate surface (151, 251) adjacent the zone (148, 248) and an opposite surface (152, 252) facing away from the zone (148, 248) exposed to external thermal influences. Further disclosed is a tissue sample receiving bag (10) comprising one or more flexible plastic cavities (12) formed from two layers of plastic sealed around their edges to form a substantially rectilinear periphery, wherein the one or more cavities (12) are within the periphery and form one or more sealable access ports (16) at one side of the periphery. A portion of the bag is left unsealed to provide a tissue sample receiving opening.)

1. A device (200) for dissociating a tissue sample into individual cells or cell masses in a closed flexible bag (10), the device comprising a mechanical dissociation mechanism (220) and a tissue sample bag receiving region (248), the device further comprising a heat transfer plate (250) for transferring thermal energy to or from the region (248), the plate having a first plate surface (251) adjacent the region (248) and an opposite surface (252) facing away from the region (248) exposed to external thermal influence, the dissociation mechanism (220) comprising a plurality of foot pedals (234, 236) each urged in a substantially linear motion towards the first plate surface solely by force from a respective resilient member (231) and each foot (234, 236) being further movable away from the first plate under the influence of the mechanical member (230, 232), the mechanical member (230, 232) is further arranged to compress the respective resilient member (231) during said movement away from the first plate.

2. The device according to claim 1, wherein the mechanism (220) comprises two or more feet (234/236) arranged to sequentially step on the tissue sample bag receiving area (28).

3. The device of claim 2, wherein the linear motion is a motion toward and away from the bag receiving area (248) in a direction substantially perpendicular to the first plate surface (151).

4. The device according to claim 2 or 3, wherein the mechanism (220) comprises two cams each having lobes arranged at 180 degrees rotational separation.

5. Apparatus according to any preceding claim, wherein when such a bag is laid flat, the feet have a common pedal area approximately equal to (up to plus or minus 30% of) the area the bag is intended to be stepped upon.

6. The device of any of claims 2 to 5, wherein the feet, when moving towards the region (248), are used to push a sample bag directly onto the adjacent first surface (251) of the heat transfer plate (250).

7. The device according to any of the preceding claims, wherein the heat transfer plate (250) has a thermal conductivity of 100W/mK or more and preferably higher than 200W/mK measured at 20 degrees celsius.

8. The device of any one of the preceding claims, wherein a final advanced position of the foot above the first surface is adjustable.

9. The device of any one of the preceding claims, wherein the mechanism (220) is within a housing (210) or substantially within a housing (210) and the tissue sample bag receiving region (248) is detachable or movable relative to the housing, e.g. by means of a hinge (255).

10. The device according to any one of the preceding claims, wherein the mechanism (220) is sealed with respect to the foot, for example by means of a flexible membrane or a sliding seal.

11. A system for cryopreserving dissociated cells, the system comprising a device (200) for dissociating a tissue sample into individual cells or cell clumps removably disposed in a controlled temperature rate modifying device such as a warmer/freezer (40), the device having one or more closed flexible bags (10) mounted or mountable therein for containing a sample for dissociation or dissociated by the device, the device comprising a mechanical dissociation mechanism (220) and a tissue sample bag receiving region (248), the device further comprising a heat transfer plate (250) for transferring thermal energy to or from the region (248), the plate having a first plate surface (251) adjacent the region (248) and an opposing surface (252) facing away from the region (248) exposed to thermal influence of the freezer (40), the dissociation mechanism (220) comprises a plurality of presser feet (234, 236) each capable of being advanced towards the first plate surface, e.g. one after the other.

12. A method for dissociating a tissue sample into cells or cell pellets, the method comprising the following steps, in any suitable order:

a) providing a tissue sample sealed or substantially sealed in a flexible sample bag (10);

b) providing a device (200), the device (200) comprising a mechanical dissociator (220), comprising a sample bag receiving region (248), and comprising a heat transfer plate (250), the heat transfer plate (250) having a first surface (251) adjacent to the region (248) and an opposite surface (252) facing away from the region (248) exposed to an external heat influence, and optionally comprising any one or more of the remaining features of the device according to the preceding claims;

c) subjecting the tissue sample to dissociation in the device (200), and

d) thermal energy is transferred into or out of the bag via the plate (250) by means of disposing the device in a controlled temperature rate changing device (40).

13. The method of claim 12, wherein step d) comprises initially introducing thermal energy into the contents of the bag via the plate (250) to assist in enzymatically dissociating or thawing the contents of the bag.

14. The method of claim 11, wherein step d) comprises removing thermal energy for cooling or for freezing the contents of the bag, and optionally comprising introducing a cryoprotectant prior to said freezing.

15. The method of any one of claims 12 to 14, wherein the dissociation device applies a cyclic pressure to the bag, for example from zero up to about 6N/cm²Or between zero and 6N/cm²Any range in between.

16. A tissue sample receiving bag when used with a device according to claims 1 to 10 or a system according to claim 11 or when used in a method according to any of claims 12 to 15.

17. A tissue sample receiving bag (10), said tissue sample receiving bag (10) comprising one or more flexible plastic cavities (12) formed with a substantially rectilinear periphery, wherein one or more cavities (12) are within said periphery and form one or more sealable or closable access ports (16) at one side of said periphery, optionally said periphery further comprising an aperture for positioning and securing said bag during pressing of said bag.

18. A tissue sample receiving bag (10), the tissue sample receiving bag (10) comprising two plastic layers sealed together around a majority of their edges to form the perimeter, and having an area of the perimeter that is not sealed to form an opening in the bag for receiving a sample into the bag, and having an additional closable opening in the form of a tube port.

19. The tissue sample receiving bag according to claim 18, further comprising a clamp for sealing said opening in use, said clamp having complementary clamping members adapted to be positioned within a sample bag receiving region (148 or 248) according to claims 1-10 or claim 11.

20. The tissue sample receiving bag according to claim 17 or 18, wherein said tissue sample receiving bag further comprises a frame (20, 20'), said frame (20, 20') having an opening (26), said opening (26) being sized to receive said one or more cavities (12), and wherein at least a portion of said perimeter overlaps said frame, said frame and said perimeter having complementary formations for retaining said at least a portion of said perimeter to said frame.

21. A tissue sample receiving bag according to claim 20, wherein said frame comprises an upper portion and a lower portion which are joined together in use, each portion further comprising a flexible cover (30), said flexible cover (30) enclosing said cavity for use as a dike around said cavity.

Technical Field

The present invention relates to an apparatus and method for dissociating tissue in an enclosed volume, and an apparatus and method for thermal control of dissociated tissue.

Background

In many fields of medicine and biology, it is desirable to take tissue samples and dissociate them into cell clumps and single cells for further processing. The number of applications is large and includes extraction of cells, for example:

a) "primary cells" can be extracted from tissues such as the liver, which can then be used in various assays commonly referred to as high throughput screening (screen);

b) tissue Infiltrating Lymphocytes (TILs) can be extracted from tumor tissue and used as a basis for autologous cell therapy;

c) the cord-shaped tissue can be used for extracting mesenchymal stromal cells;

d) tumors can be excised and their cells analyzed for "neo-antigens"; and

e) tissues can be dislocated and cells can be examined, whereby so-called cellular multinomics (e.g. proteomics, genomics, epigenomics) can be studied for many purposes including personalized medicine.

In many applications, it is desirable to maintain as many healthy cells as possible and keep them in a clean, sterile condition. In this application, closed, sterile and similar terms are intended to mean a condition whereby the biological material is separated from its surroundings, but not necessarily completely free of bioburden or other contamination, as long as it is small enough that such bioburden or other contamination (if any) has no significant effect on the viability or availability of the dissociated material.

One technique for tissue dissociation of cells is known from WO2018/130845, the content of which is incorporated herein by reference as if the wording was repeated herein. In this application, sterile tissue processing methods, kits and devices for dissociating solid tissue to derive eukaryotic cells as single cells or aggregates of small cell numbers are disclosed. The present disclosure also describes a semi-automated sterile tissue processing method. WO2018/130845 explains that the conditions during solid tissue dissociation and the time taken to harvest cells have a considerable influence on the viability and recovery of the final cellularise material. A kit is presented that together with hardware can introduce enzymes into the hanging bag to aid in dissociation, the kit comprising a separate bag into which the dissociated sample and cryoprotectant can be pumped for freezing after initial cooling.

US 643979 describes a kneading device which includes internal baffles to assist in mixing closed bags of material, but does not take into account the thermal control of this arrangement.

In this context, the inventors of the present invention have recognized that dissociating cells requires consideration of more parameters than are considered in WO2018/130845 to improve the performance of the dissociation, freezing and thawing processes, in particular thermal control during such processes not addressed in WO2018/130845 or US 643979.

Disclosure of Invention

The present invention relates to an apparatus in the form of a pedal device for efficiently dissociating tissue into individual cells or cell clumps (typically mammalian cells) and addressing the need for improved thermal control during the dissociation process. According to another aspect, the present invention relates to a thermal control method for use with the above-mentioned pedal device and the subsequent dissociated tissue processing step. According to another aspect, the present invention relates to a disposable flexible container, such as a bag, suitable for use in the above mentioned device. The above-mentioned aspects are expressed in the claims appended hereto. Further advantages and benefits of the present invention will become readily apparent to those skilled in the art, in view of the following detailed description, which provides examples of the invention.

Drawings

The invention will now be described in more detail with reference to the accompanying drawings, in which:

FIG. 1 shows a front view of a pedal device for dissociating tissue into individual cells or cell clumps within a closed sample container;

figures 2 and 3 show the device of figure 1 in two different respective operating positions;

FIG. 4 shows a plan view of the device shown in the previous figures;

FIG. 5 shows another plan view of an alternative configuration of the device;

figures 6, 7 and 8 show three different configurations of sample containers suitable for use with the device of figures 1 to 5,

FIG. 9 shows the specimen bag ready for use;

FIGS. 10, 11a, 11b and 11c show alternative ways of sealing the specimen bag;

figures 12, 13 and 14 illustrate apparatus and techniques for preparing bags for use;

FIG. 15 illustrates loading of a specimen bag or container into the depression apparatus;

fig. 16, 17 and 18 show an apparatus for separately dissociating a sample;

FIGS. 19, 20 and 21 show an apparatus for controlling the temperature of a sample or a divided sample; and

figures 23 to 25 show further embodiments of the depressing means.

Detailed Description

Referring to fig. 1, there is shown a pedal device 100 for dissociating tissue into individual cells or cell masses within a closed and at least initially sterile, generally flat-sided, relatively thin sample container bag 10. The device comprises a housing 110 formed by an assembly of parts that can be removably inserted into a temperature controlled device, such as a controlled temperature rate changing freezer, defroster or warmer, e.g. known as Via Freeze^TMOr any other device that provides a controlled rate change in temperature (shown schematically in fig. 1 and described generally herein as freezer 40). In practice, the housing will include a coverA cover (not shown). In use, the device and bag provide a closed system to dissociate tissue (e.g., a resected tumor, a portion of a resected tumor, or a needle biopsy, etc.), and then cryopreserve the resulting cell suspension for subsequent analysis without the need to pass the dissociated sample out of the bag 10.

The housing 110 has a chassis 112, a motor unit 114 is attached to the chassis 112, the motor unit 114 comprises an electric motor and a gearbox having an output speed of 10-300 rpm. The output shaft of the motor and gear box 114 has a crank 116, the crank 116 driving a connecting rod 118, the connecting rod 118 in turn being pivotably connected to a pedal mechanism 120, the pedal mechanism 120 will move through one pedal cycle for each revolution of the crank 116, i.e. a pedal cycle between 0.2 and 6 seconds. In more detail, the pedaling mechanism has a parallelogram four-bar linkage comprising two spaced pivots 122 and 124 rigidly mounted to the chassis 112 which pivotally mount two opposing parallel horizontal bars 126 and 128, respectively. Each of the horizontal bars has two parallel pedal levers 130 and 132, one pivotally connected thereto on each side of the pivots 122 and 124, together forming a parallelogram linkage. The connecting rod 118 is conveniently pivotably held to an extension of the top horizontal rod such that movement of the extension causes cyclic up and down movement (in the orientation shown) of the depressible bars 130 and 132. Foot assemblies 134 and 136 are connected to each of the foot levers 130 and 132. due to the cyclical motion mentioned above, the foot assemblies 134 and 136 will move up and down in a sequential manner with the motion of the crank 116, i.e., one foot will move down when the other is up and vice versa.

The foot assemblies 134 and 136 each include a planar base plate 138 and 140, respectively, each spring mounted to an upper foot frame 142 and 144, respectively, by coiled metal springs 146. In the arrangement described above, or an equivalent arrangement if used, the spring 146 is preloaded. In this case, the combined preload is preferably 40-80N, more preferably 30-70N, preferably about 60N for each foot. The combined spring rate is 1-5N per mm stroke, preferably about 3N per mm, and the expected foot stroke is about 8-12 mm, preferably about 10 mm. In addition, the surface area of each foot is intended toIn the range of about 20 to 50 cm²Preferably about 35 cm². This results in a nominal pressure on the bag of up to about 6N/cm at zero (when the foot is lifted off the bag or substantially unloaded) and²(about 9 psi). The preferred nominal pressure is about 2N/cm²(about 3 psi). However, a given bag may not contain a uniform material at least at the beginning of the pedaling process, and then there will be a block of material where the applied force will be concentrated and thus the pressure described as 'nominal', which is an idealized situation, such as to provide a minimum pressure resistance of the bag 10 applied towards the end of the pedaling process.

At the bottom of the chassis is a receiving area 148 for the flexible bag 10, and adjacent to the receiving area 148 is a heat transfer plate 150. Area 148 is large enough to allow sample processing bag 10 to be slid onto plate 150 via the front of the chassis (the front is shown in fig. 1). The plate includes: upper surface 151 on which bag 10 rests; and a lower surface 152 which is exposed in use for heating or cooling by external influences. The upper surface 151 is substantially parallel to the bottom plates 138 and 140 of each foot so that the bottom plates move substantially parallel to the surface 151. Stated another way, the flat bottom plate moves in a direction substantially perpendicular to surface 151, which prevents significant lateral forces on mechanism 120. The plate 150 is formed of a metal, preferably aluminum or copper or gold or silver or an alloy containing those metals. The thermal conductivity measured at 20 degrees Celsius is preferably higher than 100W/mK and more preferably higher than 200W/mK. The thickness of the plate 150 material is about 3 mm or less and provides a low thermal mass, and thus a faster response of the contents of the bag 10 to follow temperature changes on opposite sides of the plate.

With additional reference to fig. 2 and 3, the device is operated by supplying current to the motor unit 114 to drive the crank 116, in this example clockwise as shown by arrow C. The crank causes the connecting rod 118 to operate the pedal mechanism 120 described above. It will be noted that the top and bottom of the crank travel (where the greatest force is applied to the mechanism 120) coincide with the lowest position of each foot assembly 134 and 136. The foot assemblies move up and down in the direction of arrows U and D to sequentially massage the specimen bags 10, making it possible for the contents of the bags 10 to move to the side away from the respective presser foot. Since the potentially solid tissue sample in the pocket can move away from the foot rest, and because the floor 138 and 140 of each foot are spring loaded (where additional elastic travel is provided for the feet even when they are at the bottom of their travel), there is then less likelihood that the mechanism will jam when a larger tissue mass is intended to be dissociated. The sequential pedaling action also reduces the likelihood of bag 10 rupture.

Fig. 4 is a plan view of the apparatus 100 described above, but with the bag 10 not in place. In particular, the relative side-by-side positions of the foot assemblies 134 and 136 can be seen, spaced apart and having a common area viewed in plan, which is approximately equal to the area of the bag 10 when laid flat, but with utility for differences in the area of about plus or minus 10% of the area of the bag 10.

Fig. 5 shows another plan view of the device 100', the device 100' being similar in structure to the device 100 described above, but in this alternative the motor 113 of the motor unit 114 is arranged transversely to the output shaft of its gearbox 115 by using a 90 degree gearbox 115 so that the motor 113 does not protrude beyond the rear wall 111 of the device 100 '. Thus, the apparatus 100' can fit into a smaller freezer volume if desired.

During the dissociation process mentioned above, the force exerted by the foot assemblies 134 and 136 is responded to by the heat transfer plate 150. This means that the sample bag 10 is pressed against the contact surface 151 of the plate 150 during processing, providing a good surface contact between the sample bag 10 and the surface 151 of the plate, and thus an improved thermal energy transfer.

Fig. 6, 7 and 8 show different embodiments of the flexible sample bag 10 mentioned above. The bag in use slides into position in the receiving area 148 in the device 100 or 100' and is located below the two mentioned feet 134 and 136. Thus, the pouch has a substantially flat configuration of about up to 12 mm thickness, with some additional compliance in order to fit the tissue sample therein. As can be seen in fig. 6, one configuration of the bag 10 is shown as being formed from two layers of plastic material sealed only at their peripheries 14 to form a central cavity 12 and a port 16 for access into the cavity 12. The bag may be formed of EVA. In use, it is preferred that the port 16 or at least one of them is large enough to receive a sample that is cut into small pieces if necessary and passed into the bag cavity 12 by means of a syringe, i.e. about 10 mm or more in diameter. However, it is also possible to include a so-called 'zip-lock' access at the end of the bag opposite the port, so that large tissue samples can be placed into the bag and then the bag resealed. The 'zip lock' may be folded over one or more times to make a seam, held folded inside the elastic channel or by means of another clamp or clamps (not shown) to reduce the possibility of leakage. Alternatively, the pouch 10 may be opened and tissue may be added. The bag may then be heat sealed with its contents in place. The bag 10 includes an angular aperture 18 for positioning the bag in the device in use and holding it in place during depression. Although the bag 10 is illustrated with one cavity 12, it would be possible to provide a bag with more than one cavity (e.g., two, three, four or five cavities), e.g., each of the plurality of cavities is elongated and has an initially open heat-sealable end and a sealable port at its other end for introducing a reagent (such as a dissociation enzyme) and for withdrawing the dissociated sample once dissociation is complete or substantially complete.

Fig. 7 shows the bag 10 of fig. 6 mounted in the positioning frame 20 by means of pins 24 on the frame fitting into the angular apertures 18. Frame 20 is an alternative way to position and hold bag 10 in place within device 100/100'. The frame 20 comprises positioning holes 22, which positioning holes 22 cooperate with means for positioning and holding the bag in place during pressing. The frame has an inner open window 26 with a smooth circular inner edge 23 to accommodate the cavity 12 and the footholds 134 and 136 in use. Frame 20 makes it easier to load and unload bags 10 into and out of apparatus 100/100 'and 100/100'.

FIG. 8 shows an alternative frame 20' having two generally symmetrical halves each similar in construction to frame 20. Each frame half additionally has a flexible shell 30, the flexible shell 30 being molded to the frame 20' such that the two halves are joined together like a clam shell that encases the bag 10. The top and bottom flexible shells act as a dike if the inner bag 10 ruptures in use. This feature is particularly useful for infectious tissue samples.

Yet another alternative, not shown, may be employed, with a simple bag-in-bag arrangement to contain the leak. In yet another alternative, the bag may comprise a base having individual wells that are resilient (at least at room temperature) such that an aliquot of the sample may be removed without using the entire sample, for example after freezing as described below. Alternatively, the sealable bag may also be heat sealed in sections for allowing the sample to be separated.

In one example, the processing of the sample placed into the bag 10 may largely follow the steps described in WO 2018/130845. In this arrangement, the sealed pouch 10 containing tissue is suspended in an aqueous solution that may contain digestive enzymes, such as collagenase and protease, to accelerate the breakdown of the tissue introduced into the pouch via the port 16. Here, the bag is placed on the plate 150 and warmed to about 35 ℃ from, for example, an external heat source to accelerate the rate of tissue digestion. One important difference proposed here is that a single sample processing bag is employed, and that digestive enzymes can be introduced through one of the ports 16 in the bag before or during dissociation. The heat transfer plate 150 may be used to introduce thermal energy into the bag by heating the plate on its underside to provide a desired temperature in the bag for enzymatic action. This heat may conveniently come from an electrically heated warming plate or an electrical heating element in or on plate 150. The amount of dissociation will depend on many parameters, such as the size, density and elasticity of the initial tissue sample, and thus the time for dissociation and the rate of depression will vary significantly. Too long or excessively forceful stepping can result in decreased cell viability. Therefore, the motor unit speed and the disengagement cycle are important. One option to solve this problem is to time the process according to a look-up table that includes the time and output speed required to dissociate similar samples. Another option is to measure the instantaneous electrical power or energy over time required to perform the dissociation process, or to measure the force or stress exerted on the plate 150 or another part of the mechanism, and to stop after a predetermined threshold is reached to indicate that the sample has sufficiently dissociated. As the power/force/stress is reduced, the dissociation is closer to completion. Another option is to measure the light absorption through the pocket-the greater the absorption, the closer the sample is to complete dissociation. Once dissociation is complete, the contents of the bag may be transferred and the cells or other components of interest may be separated and placed back into a fresh bag for freezing in the device 100/100'. Alternatively, and preferably, the entire dissociated material may be left in the bag and device for freezing. The cryoprotectant is introduced into the bag through port 16.

Another difference between the present method and the method described in WO2018/130845 is that once the cryoprotectant is introduced, there is a device in the pouch where the dissociated sample and the cryoprotectant are installed (or retained), and the entire device is installed in the freezer 40 as described above. The base of the freezer is cold and thus draws thermal energy from the bag 10 via the heat transfer plate 150. To control the formation of ice and prevent the sample from being too cold as the bag cools, it may be massaged by feet 134 and 136 in the manner described above, albeit at a slower rate than that used for dissociation, to control ice nucleation and thus improve the viability of the cells after thawing. The motor unit 114 may be supplied with electrical energy via a wire conductor to maintain movement of the mechanism 120 within a freezer, such as the freezer 40 (fig. 1).

Cleaning after use is made easier as the device is removable from the freezer.

When use is required, the frozen dissociated sample in bag 10 can be quickly thawed in device 100/100 'by further external heating of plate 150 and/or by partially immersing device 100/100' in a warmed water bath maintained at about 37 ℃ and removing the cryoprotectant. In each case, the bag may be massaged during thawing. If the enzymes are still present, they can also be removed (if desired), for example by means of filtration. In general, they will have little or no effect on cells during cryopreservation, as their action ceases at low temperatures. All of the process manipulations, warming, dissociating, cooling, freezing, and then thawing, occur for the sample in the same sealed flexible bag 10 and may be performed in a single device. Not only is this time and space efficient, it enables a single record to capture everything that occurs for the sample during processing (e.g., temperature, duration, dissociation rate, freezing protocol) and reduces the likelihood of errors, such as the sample spending too much time in an uncontrolled environment between processing machines.

More specific examples of devices and techniques used in tissue sample processing and freezing are given below.

Fig. 9 shows an example of a pouch 10 formed from a thermoplastic material such as EVA or PVC film and having an opening 11 for receiving a tissue sample T. The bag comprises a tube 13 attached to one or more ports 16 (fig. 6), which comprises one or more branches 17, a compression valve 19 and a standard luer type connector 15. The single line shown is illustrative only-bag 10 may include additional parallel tubes connected via multiple ports 16.

Once the tissue T is inside the bag 10, the opening 11 can be sealed by a mechanical clamping seal 9, shown closed and sealed in fig. 10 and open in dotted lines in the same figure, and/or by means of heat sealing using a heat sealing machine 50 as shown in fig. 11a, to produce one or more heat-sealed closure strips (e.g. a plurality of parallel strips) 8, each method forming a sealed cavity 12 (fig. 6, 7 and 9).

An alternative or additional means for sealing the bag 10 is shown in fig. 11b and 11 c. As shown in fig. 11c, bag 10, after heat sealing at seal 8, may be clamped in a two-piece clamp 60, which clamp 60 includes a top bar 62 and a bottom bar 64 forced together by a pair of screws 66. Fig. 11b shows the clamp 60 in an exploded condition, but in use the screw 66 need not be completely removed from the rest of the clamp prior to insertion of the bag 10. The carrier rod 62 has a tapered recess 68 in which the complementary wedge formation 61 is located when clamped in position 68. The recesses and wedges concentrate the clamping force at the apex of the wedge 65, providing a higher clamping force at the apex than can be achieved by a flat clamping surface. For still greater clamping force, in the ram, the apex 65 has a small channel 67 at its peak which meets with a complementary ridge formation 69 in use. The force is sufficient to eliminate the need for heat seal 8, but for additional safety, such a seal is shown. The clamping force is further enhanced by the thickness and rigidity of the top and bottom rods, which are not easily bent, and thus maintain the clamping force applied by the screws 66. Fig. 11c shows the clamp 60 in a clamped condition. The protrusion 63 meets with features of the depression 100/100' or 200 (as described below) to inhibit movement of the clamp, and thus the clamped bag 10, during depression. The size and shape of the outer perimeter and height of the clamp 60 fit into a complementary portion of the sample receiving area 148 (or 248 of fig. 22, etc.) and thus provide further positioning of the clamped bag 10 during depression. Although not shown, the clamp 60 may also incorporate additional frames 20, 20', as shown in fig. 7 and 8, with the clamp rigidly mounted to one end of the frame and the port 16 (fig. 6 and 9) supported at the other end of the frame.

Referring to fig. 12, in use, once sealed, digestive enzymes E may be introduced into the cavity 12 via the tube 13, for example by injecting the enzymes into the bag using the syringe 5 attached to the branch connection 17. By holding the bag in an upright orientation, air can then be removed from the cavity 12 by withdrawing the plunger of the syringe 5, as shown in fig. 13. Initial mixing of enzyme E and tissue T can be performed by hand, as shown in fig. 14.

The bag 10 may then be initially loaded into the step-down device 100 for dissociation, with or without the frame 20/20' and the bank cover 30, as shown in fig. 15.

The dissociation process then occurs as described above. Once completed (which may take between a few minutes and a few hours, e.g. about 10 minutes to 7 hours, preferably 40 minutes to 1 hour), the dissociated liquefied sample may be subdivided into aliquots, e.g. using the bag sets described above, and additional sample aliquot bags 7 connected to the branch 17 as shown in fig. 16. In this case, the syringe 5 is used to draw the liquefied sample out of the bag 10 in the direction of arrow F, the valves 19a and 19b are open, and the valve 19c adjacent to the sample aliquot bag 7 is closed. Once sufficient sample is withdrawn into the syringe 5, valve 19b is closed, valve 19a remains open, and valve 19c is open. The syringe is then used to force the liquid into the sample aliquot bag 7 in the direction of arrow F in fig. 17. The tube 13 of the aliquot bag 7 may be heat sealed by means of a jig heat sealing machine 55 and is shown in fig. 18. This process can be repeated until a sufficient aliquot is obtained, or until no more sample remains. The bag may already be partly separated to make sealing each compartment easier.

As described above, the sample bag 10 may remain in the step-down device 100 (fig. 15), and the step-down device may then be loaded into a controlled rate temperature changing device, in this case a freezer 40 as shown in fig. 19. This technique allows the step to continue during freezing to inhibit ice crystal formation, but in practice the bag 10 may be removed prior to freezing and the freezer 40 then used only to cool the sample through the heat transfer plates during the step. In the alternative, the aliquot sample bag 7 may replace the entire sample bag 10. In another alternative, the freezer 40 can be used to gently cool the untreated or treated sample to about 4 degrees celsius by having the step-down device 100 mounted on top of the freezer 40 (with its lid open so the base 150 is cooled), as shown in fig. 20. In another alternative, it is possible to remove the base 150 and place the base 150 into a freezer with the freezer lid in place, as shown in fig. 21. In yet another alternative (not shown), the bag 10 or 7 may be frozen directly in the freezer 40.

The invention is not to be seen as limited by the embodiments described above, but may be varied within the scope of the appended claims, as is readily apparent to a person skilled in the art. For example, the pedaling mechanism described above is preferred because it provides a fully pivotal mechanical interconnection that is less likely to jam in cold conditions than a sliding surface, but the mechanism may be replaced with any mechanically equivalent means for pedaling two or more feet in sequence. The flat foot described may be replaced with a roller foot, where the pedaling motion is from side to side, rather than up and down. The described pedal (or its mechanical equivalent) is preferably at a rate of 2 or 3 pedals per second for each foot to optimize dissociation and maximize cell recovery, and is a steady pedal, but may be faster or slower or intermittent for different cell types.

Since the device 100/100' is intended to be placed in a freezer and subjected to extremely low temperatures (e.g., minus 80 degrees celsius or less), the use of metal parts (particularly those like the spring 146) is preferred (since the polymer part becomes much stiffer at low temperatures). Furthermore, closely fitting parts like pistons and cylinders can become stuck or not fit at very low temperatures, so a simple pivotable linkage like the described mechanism 120 is preferred.

Figures 22, 23 and 24 show an alternative depression device 200 that is similar in size and function to device 100 described above. The device 200 has certain differences, which are described in more detail below.

Referring to fig. 22, the primary difference between device 100 and device 200 is that device 200 has a different pedaling mechanism 220 than mechanism 120 of device 100. The two foot pedals 234, 236 are driven in cyclically alternating pedaling motion (similar to the motion shown in fig. 2 and 3) by a 24 volt DC electric motor 213 (fig. 23), the electric motor 213 being part of an electric motor unit 214, the electric motor unit 214 having a rotary encoder that provides feedback to a controller 221 (fig. 23) for monitoring and controlling the speed of the pedaling motion. The motor drives a cam shaft 224 via a toothed belt 222. The camshaft includes a pair of cams 230, 232 offset by 180 degrees, each contoured in this case with a cycloid shape to provide simple harmonic motion of the cam followers. Each cam is operable to move a cam follower assembly comprising an associated elastomeric follower 225, 227 which rides on the profile of the cam with the follower axle 221, 223 in force transmitting relationship with the spring follower bracket 226, 228. Each bracket 226, 228 slides in a linear guide 229 and a respective foot 234, 236 is connected to the bracket. Each assembly is in turn forced upward by a respective one of the driven wheels (as it moves away from the pedaling condition with the foot by virtue of the cam profile) as the respective cam is rotated by the motor against the urging force of the return spring 231. As the cam rotates further and the cam profile recedes, the spring 231 associated with each follower assembly forces the assembly and foot downward with a pedaling force.

Thus, the pedaling force is limited by the spring rate of the associated follower assembly spring 231 and not the power of the drive motor. 1. The force applied to the bags is limited in use by the springs, as the mechanism drives the feet upwards and the springs push them back downwards. This ensures that:

a. the motor cannot stall (regardless of the size or structure (texture) of the tumor);

b. the sample is not compressed with excessive force and the bag does not rupture;

c. the maximum pressure applied to the bag is lower than the pressure tested during bag manufacture; and

d. as described below, the hinged bag receiving area 248 can accept the sample bag and any clamps used without having to pre-position the feet. In other words, the foot may be in any position when receiving the bag because the hinged sample region 248 is closed with respect to the foot, and any sample may be compressed by the foot at that time, if desired, when the hinged region is closed with respect to the foot.

Referring also to fig. 23 and 24, the device 200 further includes a flexible sealing membrane 241 extending from the device housing 210 to the upper portions of the two feet 234, 236 that provides a fluid and dust tight seal between the sole of the foot and the remainder of the pedaling mechanism 220. This arrangement inhibits contamination of the mechanism if the compression bag cracks during use. While a membrane 241 is preferred, the feet may slide in a seal, such as a lip seal, mounted to the barrier separating the mechanism 220 from the pocket area 248 and achieving similar resistance to mechanism contamination (if desired).

The device 200 also includes a heat transfer plate 250 that performs the same function as the heat transfer plate 150. However, this plate 250 is hinged to one side of the housing at hinge 255 (fig. 24), making insertion and removal of the bag to be stepped on (as shown in fig. 6, 7 and 8) easier. The heat transfer plate 250 includes a temperature sensor 256 that allows the temperature of the plate 250 and bag receiving area 248 to be monitored and recorded by the controller for quality control. The plate 250 has a first surface 251 and a second surface 252, which have the same function as the surfaces 151 and 152 described above.

Each foot is height adjustable relative to the heat transfer plate 250 of the device 200 and an indication of its movement is also monitored by the controller. Thus, even though the rotary encoder may indicate that the motor is turning, a mechanical failure, such as a failure of the toothed belt 222, may still be detected by the controller, and appropriate action, such as sounding an alarm, may be implemented.

The device 200 has the same external dimensions as the device 100, and the device's housing 210 is intended to slide inside the controlled rate freezer 40 with the freezer lid in place, as described above and shown in fig. 21.

For convenience, the invention as illustrated in the figures is described using terms such as up, down, and more descriptive terms such as foot, pedal, and pedal, but in fact, the illustrated devices may be oriented in any manner such that those terms become, for example, inverted or less descriptive in the new orientation. Therefore, no limitations with respect to orientation should be interpreted by such terms or their equivalents.

The invention provides an apparatus (100/100') for dissociating a tissue sample into individual cells or cell clumps in a closed flexible bag (10), the apparatus comprising a mechanical dissociation mechanism (120) and a tissue sample bag receiving region (148), the apparatus further comprising a heat transfer plate (150) for transferring thermal energy to or from the region (148), the plate having a first plate surface (151) adjacent the region (148) and an opposite surface (152) facing away from the region (148) exposed to an external heat influence.