GB2586567A

GB2586567A - Closed tissue disaggregation and cryopreservation

Info

Publication number: GB2586567A
Application number: GB1902763.0A
Authority: GB
Inventors: John Morris George; Lamb Stephen
Original assignee: Asymptote Ltd
Current assignee: Asymptote Ltd
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2021-03-03
Anticipated expiration: 2039-03-01
Also published as: GB2586567B; GB201902763D0

Abstract

A device 100 for the disaggregation of tissue samples into individual cells or cell clumps in a closed flexible tissue sample bag 10. The device comprises a mechanical disaggregation mechanism 120, a bag receiving area 148 and a heat transfer plate 150 having a first plate surface 151 adjacent area 148 and an opposing surface 152 exposed to the thermal influence. The mechanical disaggregation mechanism may comprise two or more resilient feet 134/136, which tread sequentially on area 148. Also disclosed is a method for disaggregating cells using said device by placing a tissue sample in a flexible bag on area 148 and subjected to disaggregation alongside transferring heat into or out of the bag via plate 150. Further disclosed is a tissue sample receiving bag 10 comprising a flexible plastics cavity 12 formed from two layers of the plastics sealed around their edges to form a generally rectilinear periphery having a cavity 12 within the periphery. The periphery has one or more sealable access ports 16 and apertures for location and securing of the bag during treading of the bag.

Description

CLOSED TISSUE DISAGGREGATION AND CRYOPRESERVATION

TECHNICAL FIELD

The present invention relates to apparatus and methods for disaggregation of tissue in a closed volume and to apparatus and methods for thermal control of disaggregated tissue.

BACKGROUND

In many areas of medicine and biology there is a need to take tissue samples and disaggregate them 10 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" may be extracted from tissue such as liver, which can be then used in various assays commonly called high throughput screening; b) Tissue Infiltrating Lymphocytes (TIL) may be extracted from tumour tissue and used as the 15 basis for an autologous cell therapy; c) Cord tissue may be used to extract mesenchymal stromal cells; d) Tumours may be excised is their cells analysed for "neoantigen"; and e) Tissue may be dislocated and cells can be examined, whereby the so-called multi-omics of cells (e.g. proteomics, genomics, epigenomics) may be investigated for many purposes including 20 personalised medicines.

In many applications it is desirable to maintain as many healthy cells as possible, and to keep them in a clean, sterile condition. In this application closed, aseptic, sterile and like terms are intended to mean the condition whereby biological material is separated from its surroundings, but not necessarily wholly free of a bioburden or other contamination, merely free enough that such bioburden or other contamination, if any, does not have a significant influence on the viability or usability of the material which is disaggregated.

One technique of tissue disaggregation of cells is known from W02018/130845, the contents of 30 which are incorporated herein by reference, as if the wording was repeated herein. In that application, an aseptic tissue processing method, kit and device is disclosed for disaggregation of solid tissue to derive eukaryotic cells into either single cells or small cell number aggregates. The disclosure also describes a semi-automatic aseptic tissue processing method. It is explained in W02018/130845 that the conditions during solid tissue disaggregation and time taken to harvest the cells have a substantial impact on the viability and recovery of the final cellularised material. A kit is proposed, which together with hardware can introduce enzymes into a hanging bag to aid disaggregation, the kit including a separate bag into which can be pumped a disaggregated sample and a crvoprotectant for freezing after initial cooling.

US6439759 describes a kneading device which includes an internal baffle to aid mixing a closed 10 bag of materials, but the thermal control of this arrangement is not considered.

With that background the inventors of the present invention have realised that there is a need to disaggregate cells taking into account more parameters than have been considered in W02018/130845, to improve the performance of the disaggregation, freezing and thawing processes, particularly thermal control during such processes, which are not addressed in W02018/130845 or US6439759.

SUMMARY OF INVENTION

The present invention according to another aspect concerns apparatus in the form of a treading device for effective disaggregation of tissue into individual cells or cell clumps, typically mammalian cells, and addressing the need for improved thermal control during the disaggregation process.

The present invention according to another aspect concerns a thermal control method used with the above-mentioned treading device(s) as well as subsequent disaggregated tissue processing steps.

The present invention according to another aspect concerns a disposable flexible container adapted 30 for use in the devices mentioned above.

The above-mentioned aspects are represented in the claims appended herein. More advantages and benefits of the present invention will become readily apparent to the person skilled in the art in view of the detailed description below which provides examples of the invention.

DRAWINGS

The invention will now be described in more detail with reference to the appended drawings, wherein: Figure 1 shows a side view of a treading device for the disaggregation of 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 operational positions; Figure 4 shows a plan view of the device shown in the previous Figures; Figure 5 shows another plan view of an alternative construction of the device; and Figure 6,7 and 8 show three different constructions of a sample container suitable for use with the device of Figures I to 5.

DETAILED DESCRIPTION

Referring to Figure 1 there is shown a treading device 100 for the disaggregation of tissue into individual cells or cell clumps within a closed and at least initially aseptic generally flat-sided and relatively thin sample container bag 10. The device includes a housing 110 formed from an assembly of parts that can be removably inserted into a freezer, for example a commercially available freezer known as Via Freeze TM Duo, or any other freezer which provides a controlled freezing rate, and shown schematically in Figure 1 as freezer 40. In practice the housing will include a cover, which is not illustrated. In use the device and bag provide a closed system, to disaggregate tissue e.g. excised tumours, parts of excised tumours or needle biopsies etc, and to then cryopreserve the resulting cell suspension for subsequent analysis without the need to transfer the disaggregated sample out of the bag 10.

The housing 110 has a chassis 112 to which is attached a motor unit 114 which includes an electric motor and gearbox, which has an output speed of 10-300 rpm. The output shaft of the motor and 30 gearbox 114 has a crank 116 which drives a connecting rod 118, which in turn is pivotably connected to a treading mechanism 120, which will be moved through one treading cycle for each revolution of crank 116, i.e. a treading cycle between 0.2 and 6 seconds. In more detail this treading mechanism has a parallelogram four bar linkage, which includes two spaced pivots 122 and 124 rigidly mounted to the chassis 112 which pivotably mount two opposed parallel horizontal bars 126 and 128 respectively. Each of the horizontal bars has two parallel treading bars 130 and 132, 5 pivotably connected thereto one on each side of the pivots 122 and 124, together forming the parallelogram linkage. The connecting rod 118 is conveniently pivotably held to an extension of the top horizontal bar, such that moving of that extension causes cyclic up and down motion (in the orientation shown) of the treading bars 130 and 132. To each treading bar 130 and 132 is connected a foot assembly 134 and 136 which, by virtue of the above-mentioned cyclic motion, will move up 10 and down with motion of the crank 116, in a sequentially manner, i.e. when one foot is up the other will be down and vice versa.

The foot assemblies 134 and 136 each include a flat faced sole plate 138 and 140 each plate being spring-mounted to a upper foot frame 142 and 144 respectively, by coiled metal springs 146. In the arrangement described above, or an equivalent arrangement if used, the springs 146 are preloaded-. In this case the combined preload is 30-50 N for each foot preferably about 40N. The combined spring rate is 1-5 N per mm of travel, preferably about 3N per mm, and the intended foot travel is about 8-12 mm, preferably about 10mm. In addition the surface area of each foot is intended to be about 20 to 50, preferably about 35 cm'. This results in a notional pressure on the bag of between zero (when the foot lifts off the bag or has substantially no load, and up to about 6 N/cm2 (about 9 psi). The preferred notional pressure is about 2N/cm2 (about 3 psi). However, given that the bag may not, at least at the start of the treading process, contain a homogeneous material, then there will be lumps of material where the force exerted will be concentrated, and so the pressure is described as 'notional' which is the idealised situation, for example to provide a minimum pressure resistance of the bag 10 exerted toward the end of the treading process.

At the bottom of the chassis is a receiving area 148 for the flexible bag 10 and adjacent the receiving area 148 is heat transfer plate 150. The area 148 is large enough to admit the sample processing bag 10 slidable onto the plate 150 via the front of the chassis (the front being shown in Fig 1). The plate includes an upper surface 151 on which the bag 10 sits, and a lower surface 152 which in use is exposed for externally influenced heating or cooling. The upper surface 151 is generally parallel to the sole plates138 and 140 of each foot, so that the sole plates move generally parallel to the surface 151. Put another way, the flat sole plates move in a generally perpendicular direction to the surface 151, which prevents significant side forces on the mechanism 120. The plate 150 is formed from metal, preferably aluminium or copper or gold or silver, or alloys containing those metals. Heat conductance is preferably above 100 and more preferably above 200 W/m K measured at 20 degrees Celsius. The thickness of the plate 150 material is about 3mm or less and provides low thermal mass and thus a quicker reaction of the contents of the bag 10 to follow temperature changes on the opposite side of the plate.

With reference additionally to Figures 2 and 3, the device is operated by supplying electrical current to the motor unit 114, to drive the crank 116, in this example clockwise as shown by arrows C. The crank causes the connecting rod 118 to operate the above described treading mechanism 120. It will be noted that the top and bottom of the stroke of the crank, where maximum force is applied to the mechanism 120 coincides with the lowermost position of each foot assembly 134 and 136. The foot assemblies move up and down in the direction of arrows U and D to massage the sample bag 10 sequentially, such that the contents of the bag 10 have an opportunity to move to one side away from the respective treading foot. Since the potentially solid tissue samples in the bag can move away from the treading foot, and because the sole plates 138 and 140 of each foot are sprung loaded, with additional resilient travel being afforded to the feet even when they are at the bottom of their stroke, then there is less chance that the mechanism will jam when larger tissue masses are intended to be disaggregated. The sequential treading action also reduces the chances of the bag 10 rupturing.

Figure 4 is a plan view of the device 100 described above, but no bag 10 is in place in this view. In particular, the relative side-by-side positions of the foot assemblies 134 and 136 can be seen, which are spaced and have a collective area viewed in plan, which area is about equal the area of the bag 10 when laid flat, but a difference in areas of about plus or minus 10% of the area of the bag 10 has utility.

Figure 5 shows another plan view of a device 100' which is similar in construction to the device 100 30 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 the use of a 90 degree gearbox 115, so that the motor 113 does not protrude beyond a backwall 111 of the device 100'. Thus, this device 100' can fit into a smaller freezer volume if needed.

During the above-mentioned disaggregation processing, the forces exerted by the foot assemblies 134 and 136 are reacted 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 good surface contact between the sample bag 10 and the plate's surface 151, and consequently improved heat energy transfer.

Figures 6, 7 and 8 show different embodiments of the flexible sample bag 10 mentioned above. The bag in use is slid into place in the receiving area 148 in the device 100 or 100'and sits under the two feet 134 and 136 mentioned. Thus, the bag has a generally flat construction, of about up to 12mm thickness, with some additional compliance in order to fit tissue samples therein. As can seen from Figure 6 one construction of a bag 10 is shown formed from two layers of plastic material sealed only at their periphery 14 to form a central cavity 12, and ports 16 for access into the cavity 12. The bag may be formed from EVA. In use it is preferred that the ports 16, or at least one of them, is/are large enough, i.e. about 10mm in diameter or larger, to accept a sample which if necessary has been chopped into small pieces and passed into the bag cavity 12 by means of a syringe. However, it is also possible to include a so called 'zip-lock' access at the end of the bag opposite the ports, such that large tissue samples can be put into the bag and the bag is then re-sealed. The 'zip-lock' can be folded over one or more times to make a seam, held folded inside a resilient channel or by means of another clamp or clamps (not shown) to reduce the chance of leakage. The bag 10 can, as an alternative, be opened and tissue can be added. The bag can then be heat sealed with its contents in place. The bag 10 includes corner apertures 18 for locating the bag in the device in use and holding it in place during treading.

Figure 7 shows the bag 10 of Figure 6 mounted in a locating frame 20 by means of pegs 24 on the frame which fit into the corner apertures 18. The frame 20 is an alternative way of locating and holding the bag 10 in place within the device 100;100'. The frame 20 includes location holes 22 which cooperate with the device for locating and holding the bag in place during treading. The frame has an inner open window 26 with a smooth rounded inner edge 23, to accommodate the cavity 12 and treading feet 134 and 136 in use. The frame 20 makes loading and unloading of the bag 10 into and out of the device 100/100' easier.

Figure 8 shows an alternative frame 20' which has two generally symmetrical halves each similar to construction of frame 20. Each frame half has additionally a flexible shell 30 moulded to the frame 20', such that the two halves come together like a clam shell enveloping the bag 10. The top and bottom flexible shells act as a bund if the bag 10 inside ruptures in use. This feature is particularly useful for infectious tissue samples.

Yet another alternative, not shown, a simple bag-in-bag arrangement could be employed to contain leaks. In yet another alternative, the bag may include a base which has resilient (at least at room temperature) separate wells, such that aliquots of sample can be removed without using the whole sample, for example after freezing as described below. Alternatively, a sealable bag may be further heat sealed into portions for allowing the separation of the sample.

The processing of a sample put into the bag 10 can largely follow the steps described in W02018/130845. In this arrangement the sealed bag 10 containing tissue is suspended in an aqueous solution which may contain digestive enzymes such as collagenases and proteases to accelerate the breakdown of the tissue, introduced into the bag via a port 16. The bag is here placed on the plate 150 and warmed from, for example, an external heat source to approximately 35°C to accelerate the rate of tissue digestion. One important difference proposed here is that a single sample processing bag is employed, and digestive enzymes can be introduced through one of the ports 16 in the bag prior to or during disaggregation. The heat transfer plate 150 can be used to introduce heat energy into the bag by heating the plate on its underside to provide the desired temperature in the bag for enzymatic action. That heat could conveniently come from an electrically heated warming plate, or electric heating elements in or on the plate 150. The amount of disaggregation action will depend on numerous parameters, for example the size, density and elasticity of the initial tissue sample, and so the time for disaggregation and the rate of treading will vary significantly. Too long or overly vigorous treading could lead to decreased cell viability. Thus, the motor unit speed and the disaggregation period is important. One option to address this problem is to time the processing according to a look-up table which includes times and output speeds required to disaggregate similar samples. Another option is to measure the instantaneous electrical power or electrical energy over time needed to perform the disaggregation processing, or to measure the force or stress exerted on the pate 150 or another part of the mechanism, and to stop after a predetermined threshold has been reached, to indicate that the sample has been sufficiently disaggregated. As the 5 power/forces/ stresses reduce the disaggregation is closer to completion. Another option is to measure light absorbance through the bag-the greater the absorbance, the closer the sample is to complete disaggregation. Once disaggregation is complete the bag contents can be transferred, and the cells or other constituents of interest can be separated and put back into a fresh bag for freezing in the device 100/100'. Alternatively, and preferably the whole disaggregated materials can be left 10 in the bag and device for freezing. A cryoprotectant is introduced in to the bag through a port 16.

Another difference between the present methodology and that described in W02018/130845 is that once a cryoprotectant is introduced, the device with the disaggregated sample and cryoprotectant in the bag is mounted (or remains in) the device, and the whole device is mounted in the freezer 40 as described above. The base of the freezer is cold and so draws heat energy from the bag 10 via the heat transfer plate 150. To control the formation of ice and prevent supercooling of the sample while the bag it is being cooled, it can be massaged by the feet 134 and 136, in the manner described above, albeit at a slower rate than for disaggregation, to control ice nucleation and so increase the viability of the cells after thawing. Electrical energy can be supplied to the motor unit 114 via a wire conductor to maintain motion of the mechanism 120 inside the freezer, e.g. freezer 40 (Fig 1).

Since the device is removeable from the freezer, cleaning after use is made easier.

When required for use, the frozen disaggregated samples in a bag 10 can be thawed rapidly in the device 100/100' by further external heating of the plate 150, and/or by partially immersing the device 100/100' in a warmed water bath, maintained at about 37°C, and the cryoprotectant removed. In each case the bag can be massaged during thawing. If the enzymes are still present, they too can be removed if needed, for example by means of filtering. Generally, they will have had little or no effect on the cells during cryopreservation because their action is halted at low temperatures. All the process manipulations, warming, disaggregation, cooling, freezing and then thawing occur with the sample in the same sealed flexible bag 10, and may be performed in a single device. This is not only time and space efficient, but it enables a single record to capture everything that happened to the sample during processing, e.g. temperatures, durations, disaggregation speed, freezing protocol, and lessens the chance for errors, such as a sample spending too much time in an uncontrolled environment between processing machines.

The invention is not to be seen as limited by the embodiments described above, but can be varied within the scope of the appended claims as is readily apparent to the person skilled in the art. For instance, the treading mechanism described above is preferred because it provides wholly pivoting mechanical interconnections which are less likely to jam in cold conditions than sliding surfaces, but that mechanism could be replaced with any mechanically equivalent means for treading two or more feet sequentially. The flat feet described may be replaced with roller feet, where the treading motion is from side to side rather than up and down. The treading described, or its mechanical equivalent, is preferably at a rate of 2 or 3 treads for each foot per second to optimise disaggregation and maximise cell recovery, and is a steady treading, but the treading could be quicker 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 lower), the use of metal parts, particularly those parts like springs 146 is preferred since polymeric parts become much more rigid at low temperatures. Also, tightly fitting parts, like pistons and cylinders, can become jammed or ill-fitting at very low temperatures so simple pivotable linkages like the mechanism 120 described are preferred.

For convenience, terms such as upper, lower, up and down, and more descriptive terms such as feet, tread and treading have been used to described the invention shown in the drawings, but in practice, the device shown could be oriented in any manner such that those terms become for example inverted or less descriptive in that new orientation. Therefore, no limitation as to orientation should be construed by such terms or equivalent terms.

Claims

CLAIMS1. A device (100/100') for the disaggregation of tissue samples into individual cells or cell clumps in a closed flexible bag (10), the device including a mechanical disaggregation mechanism (120) and a tissue sample bag receiving area (148), said device further including a heat transfer plate (150) for transferring heat energy to or from the area (148), the plate having a first plate surface (151) adjacent the area (148) and an opposing surface (152) exposed to external thermal influence 10 which faces away from the area (148).
2. A device as claimed in claim 1 wherein the mechanism (120) is an oscillating treading mechanism and includes two or more feet (134/136) which tread sequentially a tissue sample bag receiving area (148).
3. A device as claimed in claim 2, wherein said feet treading motion is motion toward and away from the bag receiving area (148) in a direction generally perpendicular to the first plate surface (151).
4. A device as claimed in claim 2 or 3, wherein said oscillating treading mechanism (120) is a pivoting lever mechanism driven by a crank (116), in turn driven by a rotary motor unit (114), optionally the mechanism providing wholly pivoting mechanical interconnections or a combination of sliding and pivoting.
5. A device as claimed in any one of the preceding claims, wherein the feet have a collective treading area about equal (plus or minus 10%) to the area of the bag intended to be trodden, when such a bag is laid flat.
6. A device as claimed in any one of claims 2 to 5, wherein said feet when moving toward the area (148), acting to push the sample bag onto the adjacent first surface (151) of the heat transfer plate (150).
7. A device as claimed in any one of the preceding claims, wherein said heat transfer plate (150) has a heat conductance of 100 W/m K or more and preferably above 200 W/m K measured at 20 degrees Celsius.
8. A device as claimed in any one of the preceding claims, wherein said mechanical disaggregation mechanism, or part thereof, for example said feet part, is sufficiently resilient to avoid jamming in use.
9. A system for cryopreservation of disaggregated cells, the system comprising the device (100/100') of any one of claims 1 to 8 removably disposed in a controller rate freezer (40), the device having mounted or mountable therein one or more flexible bags (10) for containing samples for disaggregation or disaggregated by said device and a cryoprotectant.
10. A method for disaggregating tissue samples into cells or clumps of cells, the method comprising the following step in any suitable order:-a) providing a tissue sample sealed or substantially sealed in a flexible sample bag (10); b) providing a device (100/100') including a mechanical disaggregator (120), including a sample bag receiving area (148), and including heat transfer plate (150) having a first surface (151) adjacent the area (148) and an opposing surface (152) exposed to external thermal influence which faces away from the area (148), and optionally including any one or more of the remaining features of the device of the preceding claims; c) subjecting said tissue sample to disaggregation in the device (100,100'), and d) transferring heat energy into or out of the bag via said plate (150), optionally at the same 25 time as step c) is performed.
11. A method as claimed in claim 10, wherein step d) includes initially introducing heat energy into the bag contents via the plate (150) to aid enzymatic disaggregation or to thaw the contents of the bag, * y *
12. The method of claim 10, wherein step d) includes removing heat energy for cooling the bag contents, or for freezing the contents of the bag, by means of disposing the device in a freezer, for example a controlled freezing rate freezer, optionally including the introduction of a cryoprotectant prior to said freezing.
13. A method as claimed in any one of claims 10 to 12, wherein said disaggregation device exerts a cyclic pressure on the bag, for example from zero to up to about 6N/cm2 or any range between zero and 6N/cm'.
14. A tissue sample receiving bag when used with the device of claims 1 to 8 or the system of claim 9, or when used in the method of any one of claims 10 to 13.
15. A tissue sample receiving bag (10) comprising a flexible plastics cavity (12) formed with a generally rectilinear periphery with the cavity (12) within the periphery, and at one side of the 15 periphery is formed one or more sealable access ports (16), the periphery also including apertures for location and securing of the bag during treading of the bag
16. A tissue sample receiving bag (10) comprising two plastics layers sealed around their edges to form plastics said periphery.
17. The tissue sample receiving bag as claimed in claim 15 or 16, further including a frame (20,20') having an opening (26) of a size that accepts the cavity (12) and wherein at least a portion of the periphery overlaps the frame, the frame and periphery having complementary formations for holding the periphery to the frame.
18. A tissue sample receiving bag as claimed in claim 17, wherein the frame includes upper and lower portions that come together in use, each portion further including a flexible cover (30) encapsulating the cavity for acting as a bund around the cavity.