Cryopreservation of biological cells and tissues

Abstract

The method involves placing an oocyte cell in a cell holder (1), securing the cell holder in a treatment station, applying a treatment solution to the cell by washing the cell with the solution, and rapidly cooling the cell holder and cell to a predetermined cryopreservation temperature for cryopreservation of the cell. The cell is cooled at a high rate sufficient to permit vitrification of the cell and any surrounding treatment solution to occur. The cell is then maintained at or below a predetermined storage temperature for storage. The method allows multiple cells to be treated simultaneously each secured within a respective cell holder.

Claims

1. An organic cell cryopreservation apparatus for use in the vitrification of at least one organic cell, said apparatus including: a plurality of cell holders, each having a filter to retain one of the at least one organic cell while allowing for fluid to pass from the cell holder; a securing frame for securing each cell holder in a predetermined orientation, the securing frame comprising: an array of cell holder receiving portions, each configured to receive one of the cell holders; and at least one securing formation for securing the cell holders in the cell holder receiving portions; and a treatment station for washing said at least one organic cell, said treatment station including: a temperature controller; at least one pipette to dispense a predetermined measure of treatment solution into each cell holder; and a docking formation for engaging a corresponding docking formation on the securing frame thereby aligning said securing frame into a predetermined orientation with said treatment station.

2. An apparatus according to claim 1, said apparatus further including a cooling station for cooling said cells to a cryopreservation temperature.

3. An apparatus according to claim 2, wherein said cooling station includes a liquid nitrogen cooled block.

4. An apparatus according to claim 2, wherein the treatment station comprises the cooling station.

5. An apparatus according to claim 1, the treatment station further comprising a drain for receiving treatment fluid draining from said cell holder.

6. An apparatus according to claim 1, said frame having a drainage path for directing treatment fluid passing through said filtration arrangement and from said cell holder to a drainage outlet.

7. An apparatus according to claim 1, wherein said filtration arrangement includes at least one of: a mesh; a membrane; a fibrous material; and a perforated section.

8. An apparatus according to claim 1, wherein the cell-holder comprises a cup-shaped holding vessel.

9. An apparatus according to claim 8, wherein the cell-holder comprises converging sidewalls.

10. An apparatus according to claim 1, wherein the treatment station comprises a multi-headed pipette to simultaneously dispense predetermined measures of treatment solution into the plurality of cell holders.

11. An apparatus for vitrification of organic cells, the apparatus comprising: a plurality of cell holders, each cell holder comprising a cup-shaped vessel with converging sidewalls and a filter for retaining an organic cell in the cell holder while allowing for fluid to pass from the cell holder; a securing frame defining an array of cell holder receiving portions and comprising at least one securing formation for securing the cell holders in the cell holder receiving portions; and a treatment station comprising a docking formation configured to hold the securing frame in a predetermined orientation relative to the treatment station, and at least one pipette for dispensing a predetermined measure of treatment solution into each cell holder.

12. An apparatus according to claim 11, wherein the treatment station comprises a multi-headed pipette to simultaneously dispense predetermined measures of treatment solution into each of the plurality of cell holders.

13. An apparatus according to claim 11, said apparatus further including a cooling station for cooling said cells to a cryopreservation temperature.

14. An apparatus according to claim 13, wherein said cooling station includes a liquid nitrogen cooled block.

15. An apparatus according to claim 13, wherein the treatment station comprises the cooling station.

16. An apparatus according to claim 11, wherein the treatment station is automated to dispense predetermined measures of treatment solution into the cell holders secured in the securing frame and drain at least part of the treatment solution away from the cell holders.

17. An apparatus according to claim 11, wherein the treatment station further comprises a temperature controller.

18. An apparatus according to claim 1, wherein the treatment station is automated to dispense predetermined measures of treatment solution into the cell holders secured in the securing frame and drain at least part of the treatment solution away from the cell holders.

19. An apparatus according to claim 1, wherein the treatment station further comprises a temperature controller.

20. An apparatus according to claim 2, wherein liquid nitrogen is used in the cooling station for cooling said cells to a cryopreservation temperature.

21. An apparatus according to claim 13, wherein liquid nitrogen is used in the cooling station for cooling said cells to a cryopreservation temperature.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

(2) FIG. 1a is a perspective view of a cell holder according to the invention;

(3) FIG. 1b is a perspective view of the cell holder shown in FIG. 1a;

(4) FIG. 1c is a detailed part perspective view of a cell holding vessel of the cell holder shown in FIG. 1a;

(5) FIGS. 1d and 1e are detailed part perspective views of alternative cell holders according to the invention;

(6) FIG. 1f is a perspective view of a storing straw according to the invention;

(7) FIG. 2a is a perspective view of a securing tray according to the invention;

(8) FIG. 2b is a detailed part top perspective view of a cell holder located in the securing tray shown in FIG. 2a;

(9) FIG. 2c is a detailed part bottom perspective view of a cell holder in the securing tray shown in FIG. 2a;

(10) FIG. 2d is a detailed part top perspective view of a cell holder in the securing tray shown in FIG. 2a;

(11) FIG. 3a is a perspective view of a cover for the securing tray shown in FIG. 2a, according to the invention;

(12) FIG. 3b is an exploded perspective view of the cover for the securing tray shown in FIG. 3a and the securing tray shown in FIG. 2a;

(13) FIG. 3c is an exploded perspective view of the cover for the securing tray shown in FIG. 3a and the securing tray shown in FIG. 2a;

(14) FIG. 4a is a perspective view of the cover for the securing tray shown in FIG. 3a mounted on the securing tray shown in FIG. 2a;

(15) FIG. 4b is an underside perspective view of the cover for the securing tray shown in FIG. 3a mounted on the securing tray shown in FIG. 2a;

(16) FIG. 4c is a perspective view of a washing station according to the invention;

(17) FIG. 5a is a detailed part perspective view of FIG. 4a;

(18) FIG. 5b is a detailed cross-sectional perspective view of the washing station shown in FIG. 4c and the tray and cover assembly shown in FIG. 4a;

(19) FIG. 6a is an exploded perspective view of the tray and cover assembly shown in FIG. 4a and a cooling station in accordance with the invention;

(20) FIG. 6b is a perspective view of the tray and cover assembly shown in FIG. 4a mounted on the cooling station shown in FIG. 6a;

(21) FIG. 7a is a perspective view of the tray shown in FIG. 2a mounted on the cooling station shown in FIG. 6a;

(22) FIG. 7b is a perspective view of the tray shown in FIG. 2a mounted on the cooling station shown in FIG. 6a with a cell holder removed;

(23) FIG. 8a is a perspective view of the cell holder of FIG. 1a and straw of FIG. 1f;

(24) FIG. 8b is a perspective view of the cell holder of FIG. 1a partially inserted into the straw of FIG. 1f; and

(25) FIG. 8c is a perspective view of the cell holder of FIG. 1a full inserted into the straw of FIG. 1f and ready for long term cold storage.

PREFERRED EMBODIMENTS OF THE INVENTION

(26) Referring to the drawings, the invention provides for a method and apparatus for the cryopreservation of oocyte, embryos and other cell types.

(27) For the sake of simplicity, the preferred embodiment will be described with reference to oocyte cells however; the invention is not limited to the cryopreservation of oocytes and is intended to be equally applicable to other biological cells and tissues including individual cells such as stem cells, groups of cells such as embryos and tissue samples.

(28) Additionally while the method and apparatus are described with reference to vitrification cryopreservation, it will be appreciate that it may be equally applied to other, methods of cryopreservation.

(29) In its simplest form the method involves placing an oocyte cell in a cell holder, securing the cell holder in a treatment station, applying a treatment solution to said cell by washing the cell with the solution, and rapidly cooling the cell holder and cell to a predetermined cryopreservation temperature for cryopreservation of the cell. Preferably the cell is cooled at a high rate sufficient to permit solidification of the cell and any surrounding treatment solution to occur. More preferably, solidification includes the substantial vitrification of the cell and surrounding fluid. The cell is then maintained at or below a predetermined storage temperature for storage.

(30) The invention includes multiple components designed to function together, enabling simple, safe and hygienic handling of the delicate cell. The method and apparatus also lends itself to multiplexing so that an individual technician may process more than one cell holder and oocyte cell simultaneously.

(31) Referring to FIG. 1a the cell holder 1 includes retaining means to retain the organic cell whilst allowing for fluid to pass over the cell and through the cell holder. The retaining means includes a filter 2 to provide a physical barrier to the cell whilst allowing passage of a treatment solution. The filter can be seen in FIG. 1c which provides a close up view of the holder.

(32) In this embodiment the holder also includes sidewalls 3, defining a cup-shaped holding vessel 4 having an open top inlet 5. In this way, in the embodiment shown in FIG. 1c, the filter 2 forms the bottom surface of the vessel. In other embodiments however, the filter may form any portion of the vessel including the bottom and/or sidewalls. The filter 2 may be a mesh, membrane, fibrous material, perforated material or otherwise porous material which allows passage of liquids but which retains the oocyte within the vessel.

(33) A further embodiment of vessel having converging sidewalls 6 is shown in detailed view FIG. 1d. The converging sidewalls reduce the likelihood of the cell being washed out of the vessel through the open top. In this view an embryo 7 is also shown. A further embodiment of the vessel holder is shown in FIG. 1e. In this embodiment, the sidewalls include cut outs.

(34) In a further embodiment not shown in the Figures the vessel may include a removable cover. The cover may include a further filter material over a fluid inlet to allow passage of fluid into the cell holder whilst preventing the cell from escaping. Thus the cell is secured in a holding chamber. The filter on the cover may be of any filter material as previously described.

(35) All of the embodiments shown in the Figures include an elongate handle 8 attached to the cell holder 1 to enable it and oocyte cell to be manually moved without direct contact from the operator. The handle also provides a convenient labelling area 9 for labelling and identification.

(36) In this embodiment, the vessel is designed to fit into an elongate tubular sheath or straw 10 as shown in FIG. 1f. The straw 10 is open at one end 11 and sealed at the other 12. A circumferential ridge 13 on the handle of the cell holder 1 engages and seals with the inner bore 14 of the tubular straw 10 thereby sealing the oocyte within the straw once the freezing process is complete.

(37) In alternative embodiments not shown in the drawings, the handle is selectively detachable. In further embodiments the handle is a separate tool designed to selectively engage and grip the cell holder so it can be moved as required. The tool may be in the form of a pincer type tool or include a simple fixed yoke or engagement formation to generally encircle and engage the cell holder. In such embodiments, the cell holder may include an engagement portion, formation or tab designed to releasably engage with the handle or tool.

(38) In still further embodiments, the cell holder includes multiple holding vessels 4 joined together to provide for treatment and cryopreservation of multiple cells. The vessels, otherwise similar to any one or more of those described above, may be positioned at a predetermined spacing from one another in order to engage with other components, as will be appreciated from the description below. Alternatively, each cell holder may include linking formations to allow for multiple cell holders to be linked together, in effect forming a multiple vessel cell holder. This way cell holders can be quickly and easily joined together in the lab to provide for as many holding vessels as required. Preferably, the linking formations are configured to position the cell holders in a predetermined spacing.

(39) The invention further includes a securing frame, for securing the cell holder. The frame is shown in FIG. 2a and in this embodiment is in the form of a tray 20. The tray includes an engagement formation 21 and a cell holder-receiving portion 22, each shown in detail FIGS. 2b and 2d, respectively. As will be appreciated with reference to the figures, in this embodiment the engagement formation 21 includes a shallow elongate locating groove for receiving and positively locating the handle 8 of the cell holder 1. In this way, the complementary shape and configuration of the cell holder handle 8 and the corresponding groove on the frame, bias the cell holder into a particular orientation and position with respect to the frame 20.

(40) In position, as shown in the detailed upper view FIGS. 2b and 2d and lower view FIG. 2c, the cell holder is positioned within or over the receiving portion 22 of the frame 20. In this embodiment, as can clearly be seen in FIG. 2a, the receiving portion 22 includes an aperture in the tray so that the cell holder is accessible from above and the bottom of the cell holder is clear or protruding from the tray bottom.

(41) The tray 20 may include a plurality of grooves for receiving a plurality of cell holders in an array. The tray illustrated in the figures has grooves for eight cell holders. However, it will be appreciated that a tray may accommodate more or fewer than eight cell holders.

(42) In the alternative configuration where the cell holder does not include a handle, the engagement formation 21 on the tray 20 may include projections which engage the outer sidewall of the cell holder. In still further embodiments, the cell holder includes corresponding engagement formations to engage the engagement formations on the tray.

(43) Returning to the figures, the invention also includes securing means for locking the cell holders into the tray. In some embodiments, the cell holder and tray may include snap locking securing formations 23 to individually lock each cell holder directly to the tray. Such securing formation can be seen with reference to FIG. 2b and engage the cell holder handle 3.

(44) In this embodiment, the securing means also includes a tray cover 24 to jointly hold the cell holders within the locating grooves 21 on the tray 20 thereby preventing their extraction and any relative movement within the tray. The cover 24 includes releasable locking means for locking to corresponding releasable locking means on the tray thereby securing the cover to the tray. The cover also includes a funnel formation and aperture 25. The funnel formation and aperture are configured such that when the cover is locked to the tray, the funnel and aperture are aligned with the open top of the cell holder. The function of the funnel will be detailed later. The cover may also include location formations 26 to engage and secure the cell holder handle. These location formations can be seen in the underside view of the cover in FIG. 3a.

(45) It will be appreciated that locked into the tray, a plurality of cell holders may be safely and easily moved by an individual technician or by a mechanical manipulating device, automated robot or the like. In addition, because the relative position of the cell holders and tray are set, the tray can be used as a guide for aligning the array of cell holders for treatment at a treatment station.

(46) In this regard the invention includes at least one treatment station to which the tray can be selectively docked and various operations performed. The station is configured for receiving the tray and includes docking formations for docking with corresponding docking formations on the tray.

(47) A first treatment station is a washing station 30 shown in FIG. 4c. The washing station includes a drain 31 for capturing waste fluid and temperature control means 32. At this station the cells are held at a predetermined temperature by the temperature control means and washed with treatment solution.

(48) A second treatment station is a cooling station 40, shown in FIGS. 6a and 6b where the cells and cell holders are cooled to below a cryopreservation temperature. Specific cooling properties of the cooling station will depend on the intended method of cryopreservation whether by slower rate freezing or rapid vitrification. The cooling station 41 includes a cooling block or similar device held at a very low temperature by liquid nitrogen.

(49) In this embodiment the treatment and cooling stations are at distinct locations and the tray transferred between. However it is also possible that the tray is held at a fixed point and the stations are moved to align with the tray as required.

(50) In use, an oocyte to be cryopreserved is placed into the holding vessel 4 of a cell holder 1 as shown in FIG. 1a. The cell holder 1 itself is generally pre-labelled with identification details regarding the cell and/or donor/s. The holder 1 is then placed into securing tray 20 so that the cell holder handle lies in the corresponding groove and the holder vessel is located in or above the receiving aperture 22. This can be seen in FIG. 2c where the base of the cell holder protrudes below the bottom surface of the tray. FIG. 2b shows the cell holder handle engaged in the locating groove and secured with snap locking securing formations 23. In the embodiment depicted, eight such cell holders are placed in the tray in an array of respective locating grooves.

(51) With the cell holders in place, the tray cover is aligned with and locked onto the tray. In position, the funnel formations and apertures on the tray cover align with the open top of the cell holding vessels.

(52) The tray is then transferred to the first treatment station, the washing station 30. Corresponding docking formations on the tray and washing station position the tray so that the vessel holders located are above the drain, adjacent the heating block 32. This can be seen in FIG. 5b where the cover 24 and funnel are positioned above the open top of the holding vessel, which is over the drain 31 of the washing station.

(53) As seen in FIG. 5a fluid dispensing pipette 50 is positioned above the funnel formation to dispense a predetermined measure of treatment solution into each funnel aperture. The fluid flows down through the funnel, out the funnel aperture and into the holder vessel passing over and washing the oocyte cell. Excess fluid drains from the vessel through the filter 2 and passes into the drain.

(54) The flow rate of fluid from the pipette and the proportions of the funnel are carefully chosen so that the treatment fluid delicately flows into the vessel. If the fluid flow rate is too high the fluid can damage the cells and/or overflow the vessel and possibly dislodge the cells. As a preventative measure, in some embodiments, an additional filtration membrane may be placed over the open top inlet 5 to retain the cell within the vessel.

(55) Furthermore, in order to encourage flow of the treatment solution through the filter, the drain 31 is held at a negative pressure. In this regard the treatment station also includes vacuum means to apply a gentle negative pressure to the drain. A vacuum pump and corresponding control electronics are positioned within a cavity 33 of the washing station.

(56) It also is critical during this process to maintain the oocyte cell at the correct temperature. To this end, the treatment fluid is held at a predetermined temperature in the pipette and the heating block 32 maintains the temperature of the cells at a predetermined level.

(57) The cell may be treated with a sequence of different treatment solutions as required. The same pipette may be used for each solution or if the solutions are incompatible, a different pipette may be used for each solution. The last treatment solution to be applied is a vitrification fluid, which is vitrified along with the cell during the cooling and solidification.

(58) In addition, it is intended that the array of oocytes in their respective holders located in the tray are all treated simultaneously. Accordingly a multi-headed pipette 50 as shown in FIG. 5a is used to dispense the treatment solution to each cell holder at the same time. Furthermore, the system also lends itself to automation or semi-automation. Since the position of the cell holders is dictated by the position of the tray, the entire washing procedure can be multiplexed and automated.

(59) As such an automated system allows precise control over the delivery of the treatment solution, the temperature of the fluid and cell, which hitherto were difficult to achieve in a manual system.

(60) Once the oocytes have been treated, the tray is removed from the washing station and transferred without delay to the cooling station 40. As with the washing station, the cooling station includes docking formations that engage corresponding docking formation on the tray 20. In position, the cell holder vessels are aligned with the cooling station vitrification block 41. The vitrification block is cooled with liquid nitrogen such that cooling occurs within moments of contact at a rate sufficient to transform the cell and any surrounding vitrification fluid into a vitrified state. The block may be immersed in or contain the liquid nitrogen so that it is at a similar temperature to the liquid nitrogen.

(61) While vitrification of the oocytes or cells occurs very quickly, the cell holders are kept in position in the cooling station for a predetermined period to ensure the cells are cooled to a suitably low temperature. With the tray mounted to the cooling station, FIG. 7a, the tray cover is removed and each cell holder containing a vitrified oocyte is removed from the tray, FIG. 7b, and inserted into a pre-cooled straw 10, FIG. 8b, or other suitable holding device. In this form the oocyte is securely contained, FIG. 8c, and can be placed into long-term cold storage.

(62) Again, the process of removal from the tray, insertion into a straw and then storage is automated. In alternative embodiments it can be performed manually.

(63) Advantageously, it will be appreciated that the system allows the preparation of the embryo/oocyte for cryopreservation to be carefully controlled. In addition, the proposed system allows for the preparation and vitrification of multiple embryos/oocytes simultaneously.

(64) The process allows for the treatment process to be uniformly controlled removing variances of the washing and vitrification process. In addition, the invention provides for less manipulation of the cells because they remain generally stationary in the cell holder through out the treatment and vitrification process. In these and other respects, the invention represents a practical and commercially significant improvement over the prior art.

(65) Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.