Methods and apparatus for washing cryopreserved cells

Abstract

A method for washing cryopreserved cells includes maintaining a receptacle with a first liquid containing cells that had been cryopreserved in a first orientation, and subsequently placing the receptacle in a second orientation. The method also includes aspirating the first liquid in the receptacle while leaving the cells in the receptacle; dispensing a second liquid into the receptacle; and aspirating the second liquid in the receptacle while leaving the cells in the receptacle. An apparatus for washing cryopreserved cells and a computer-readable storage medium storing instructions for washing cryopreserved cells are also disclosed.

Description

Methods and Apparatus for Washing Cryopreserved CellsRELATED APPLICATION

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application Serial No. 63 / 595,749, filed November 2, 2023, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] This application relates to methods, devices, and apparatus for washing cryopreserved cells.BACKGROUND

[0003] Separation of biological cells is a critical step in many biological processes and assays. Conventional methods for separating biological cells include subjecting the biological cells in a liquid to a large acceleration, such as by using a centrifuge. The centrifugal force causes the biological cells to travel to the bottom of a tube and form a precipitate, allowing a remaining liquid called supernatant to be separated from the precipitate.

[0004] However, the mechanical force applied on the biological cells may affect the biological cells. For example, too much centrifugal force will cause lysis of biological cells.SUMMARY

[0005] Accordingly, there is need for methods, devices, and apparatus that do not require large acceleration for separating biological cells. Such methods, devices, and apparatus plates may replace the conventional methods, devices, and apparatus for separating biological cells. Such methods, devices, and apparatus may better retain physical and biological properties of cells during separation by reducing or eliminating the need for applying large acceleration or force, thereby enhancing the accuracy or efficiency of biological processes and assays.

[0006] This is especially important for cryopreserved cells, which often require a large volume of liquid for thawing or post-thawing steps. Conventional methods for separating cells in a large volume of liquid require the use of a centrifuge. In addition, additional washing of the separated cells also requires the use of a centrifuge. Thus, cryopreserved cells are often subjected to centrifugation multiple times, which increases the chance of damages to such cells.

[0007] A number of embodiments that overcome the limitations and disadvantages of existing methods, devices, and apparatus are presented in more detail below. These embodiments provide methods, devices, and apparatus for separating biological cells.

[0008] As described in more detail below, in accordance with some embodiments, a method includes maintaining a receptacle with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; and, subsequent to maintaining the receptacle in the first orientation for the first period of time, placing the receptacle in a second orientation distinct from the first orientation. The second orientation has a second angle distinct from the first angle with respect to the vertical axis. The method also includes aspirating the first liquid in the receptacle while leaving the cells in the receptacle; dispensing a second liquid into the receptacle; and aspirating the second liquid in the receptacle while leaving the cells in the receptacle.

[0009] In accordance with some embodiments, an apparatus includes a receptacle holder for holding a receptacle; a tilting device coupled with the receptacle holder for placing the receptacle holder in a first orientation at a first time and placing the receptacle holder in a second orientation distinct from the first orientation at a second time distinct from the first time; one or more liquid handling devices; one or more processors; and memory storing one or more programs for execution by the one or more processors. The one or more programs include instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle in a second orientation distinct from the first orientation; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the cells in the receptacle. The second orientation has a second angle distinct from the first angle with respect to the vertical axis.

[0010] In accordance with some embodiments, a method includes computer-readable storage medium storing one or more programs for execution by one or more processors in communication with a tilting device and one or more liquid handling devices. The one or more programs including instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having afirst angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle holder in a second orientation distinct from the first orientation; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the cells in the receptacle. The second orientation has a second angle distinct from the first angle with respect to the vertical axis.

[0011] Such methods, apparatus, and computer readable storage medium may replace conventional methods, apparatus, and computer readable storage medium. In some cases, such methods, apparatus, and computer readable storage medium may complement conventional methods, apparatus, and computer readable storage medium.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] For a better understanding of the aforementioned embodiments as well as additional embodiments, reference should be made to the Description of Embodiments below, in conjunction with the following drawings in which like reference numerals refer to corresponding parts throughout the figures.

[0013] Figure l is a perspective view of a plate in accordance with some embodiments.

[0014] Figure 2 is a cross-sectional view of the plate shown in Figure 1.

[0015] Figure 3 illustrates a tube in accordance with some embodiments.

[0016] Figures 4A-4M illustrate devices for separating biological cells or one or more portions thereof in accordance with some embodiments.

[0017] Figure 5 illustrates experimental results obtained by using the methods described herein.

[0018] Like reference numerals refer to corresponding parts throughout the drawings.

[0019] Drawings are not necessarily drawn to scale unless indicated otherwise.DESCRIPTION OF EMBODIMENTS

[0020] Methods, devices, and apparatus for washing samples are described. Reference will be made to certain embodiments, examples of which are illustrated in the accompanyingdrawings. While the claims will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the claims to these particular embodiments alone. On the contrary, the embodiments are intended to cover alternatives, modifications and equivalents that are within the spirit and scope of the appended claims.

[0021] Moreover, in the following description, numerous specific details are set forth to provide a thorough understanding of the embodiments. However, it will be apparent to one of ordinary skill in the art that the embodiments may be practiced without these particular details. In other instances, methods, procedures, components, and networks that are well- known to those of ordinary skill in the art are not described in detail to avoid obscuring aspects of the embodiments.

[0022] It will also be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first channel could be termed a second channel, and, similarly, a second channel could be termed a first channel, without departing from the scope of the embodiments. The first channel and the second channel are both channels, but they are not the same channel. Similarly, a first period of time could be termed a second period of time, and, similarly, a second period of time could be termed a first period of time, without departing from the scope of the embodiments. The first period of time and the second period of time are both periods of time, but they are not the same period of time.

[0023] The terminology used in the description of the embodiments herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the embodiments and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0024] Figure 1 is a perspective view of a receptacle (e.g., plate 100) in accordance with some embodiments. The plate 100 has atop surface 120 and abottom surface 130 opposite to the top surface 120. A plurality of wells 112 (e.g., wells 112-1 through 112-8) is defined in the plate 100. The plate 100 includes a first portion 140 that corresponds to a bottom of theplurality of wells 112 and a second portion 150 that corresponds to one or more walls of the plurality of wells 112. In some embodiments, the plate 100 is formed integrally. In some embodiments, the plate 100 is formed by attaching two or more portions together (e.g., by bonding separately formed first and second portions 140 and 150). In some embodiments, as shown in Figure 1, the plurality of wells 112 is arranged in an array (e.g., 2-by-3 array, 2-by-4 array, 3-by-4 array, 4-by-6 array, 6-by-8 array, 8-by-12 array, 16-by-24 array, 32-by-48 array, etc.). In some embodiments, a respective well 112 is a cylindrical well (e.g., a cross-section of the respective well 112 along a plane substantially parallel to the plate 100 has a shape of a circle). Also shown in Figure 1 is line II-II, from which the cross-sectional view of Figure 2 is taken.

[0025] Figure 2 is a cross-sectional view of the plate 100 shown in Figure 1. In some embodiments, a respective well 112 has a width (W) of 2 mm - 170 mm (e.g., 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 75 mm, 80 mm, 85 mm, 90 mm, 95 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, or any interval between two of the aforementioned values, such as 5 mm - 8 mm). In some embodiments, the respective well 112 has a height (H) of 2 mm - 170 mm (e.g., 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 75 mm, 80 mm, 85 mm, 90 mm, 95 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, or any interval between two of the aforementioned values, such as 3 mm - 70 mm).

[0026] Figure 2 also shows a first axis 202 defined by a first channel (e.g., well 112-1) and a second axis 204 defined by a second channel (e.g., well 112-2). In some embodiments, the first axis 202 is parallel to a longitudinal direction of the first channel (e.g., well 112-1). In some embodiments, the first axis 202 passes through a center of the first channel. In some embodiments, the second axis 204 is parallel to a longitudinal direction of the second channel (e.g., well 112-2). In some embodiments, the second axis 204 passes through a center of the second channel. In some embodiments, the first axis 202 is parallel to a height-wise direction of the first channel (e.g., well 112-1). In some embodiments, the second axis 204 is parallel to a height-wise direction of the second channel (e.g., well 112-2). In some embodiments, the first axis 202 is parallel to the second axis 204. In some embodiments, the first axis 202 is nonparallel to the second axis 204 (e.g., the first axis 202 is at a predefined non-zero angle fromthe second axis 204, such as 1 degree, 5 degrees, 10 degrees, 15 degrees, 20 degrees, or between any two angles selected from the aforementioned angles).

[0027] Although Figures 1 and 2 illustrate a plate with a plurality of wells, a receptacle having only a single well or channel (e.g., a tube) may be used instead, or in addition.

[0028] Figure 3 illustrates a tube 300 in accordance with some embodiments. The tube 300 includes a tube wall 310, which defines a channel 302. The channel 302 has, or defines, an axis 318. In some embodiments, the axis 318 is parallel to the longitudinal direction of the channel 302.

[0029] Figure 3 also illustrates that the tube 300 includes a solution 320 containing biological cells 330 or particles in some cases. However, as a person having ordinary skill in the art would recognize, the solution 320 and the biological cells 330 are not part of the tube 300.

[0030] Figures 4A-4M illustrate an apparatus 400 for separating biological cells or one or more portions thereof in accordance with some embodiments.

[0031] In Figure 4A, the apparatus includes a receptacle holder 410 and a tilting device 420. In some embodiments, the apparatus also includes an impulse device 430.

[0032] As shown in Figure 4A, in operation, a receptacle 300 (e.g., a tube) containing a solution 320 with biological cells 330 or particles may be placed in the receptacle holder 410.

[0033] In some embodiments, the receptacle holder 410 is in a second orientation. In some embodiments, the second orientation is a substantially vertical orientation as shown in Figure 4A. In some embodiments, the receptacle holder 410 is deemed to be in a substantially vertical orientation when a channel defined by a receptacle to be held by the receptacle holder 410 defines an axis that is in a substantially vertical orientation. In some embodiments, the receptacle holder 410 (e.g., a tube holder) is deemed to be in a substantially vertical orientation when a channel defined by the receptacle holder 410 (e.g., a channel into which the tube is to be inserted) defines an axis that is in a substantially vertical orientation. For example, the first axis 318 defined by the channel 302 of the receptacle 300 or a channel of the receptacle holder 410 is substantially vertical (e.g., the axis 318 is substantially parallel to a vertical direction 440). In some embodiments, the receptacle holder 410 (e.g., a plate holder) is deemed to be in a substantially vertical orientation when a base surface of the receptacle holder 410 is substantially perpendicular to the vertical direction 440.

[0034] Figure 4A also shows one or more processors 412 and memory 414 storing one or more programs for execution by the one or more processors 412. In some embodiments, the one or more programs include instructions that cause the one or more processors to send one or more signals or instructions to devices in communications with the one or more processors (e.g., the tilting device 420, an aspirator, a dispenser, etc.).

[0035] In addition, Figure 4A shows that the receptacle holder 410 holder is to be rotated or tilted by the tilting device 420.

[0036] Figure 4B illustrates that the receptacle holder 410 is a first orientation. In some embodiments, the first orientation is a non-vertical orientation as shown in Figure 4B. In some embodiments, the receptacle holder 410 is deemed to be in a non-vertical orientation when the channel defined by the receptacle to be held by the receptacle holder 410 defines an axis that is in a non-vertical orientation. In some embodiments, the receptacle holder 410 (e.g., a tube holder) is deemed to be in a non-vertical orientation when a channel defined by the receptacle holder 410 (e.g., a channel into which the tube is to be inserted) defines an axis that is in a non- vertical orientation (e.g., the axis has a non-zero angle with respect to the vertical direction). For example, the first axis 318 defined by the channel 302 of the receptacle 300 or a channel of the receptacle holder 410 is non-vertical (e.g., the axis 318 is non-parallel to the vertical direction 440). In some embodiments, the receptacle holder 410 (e.g., a plate holder) is deemed to be in a non-vertical orientation when a base surface of the receptacle holder 410 is nonperpendicular to the vertical direction 440.

[0037] Figure 4C shows that the receptacle holder 410 is maintained in the first orientation. The tilting of the channel defined by the receptacle 310 speeds up settling of the biological cells or particles.

[0038] Figure 4C also shows that the receptacle holder 410 is to be rotated or tilted back by the tilting device 420.

[0039] Figure 4D illustrates that the receptacle holder 410 is in the second orientation. In Figure 4D, the biological cells 330 or particles are settled down to a bottom of the receptacle. In some cases, because the biological cells 330 or particles settled while the receptacle was in a tilted orientation, the biological cells 330 or particles may have settled in a non-symmetric manner (e.g., the biological cells 330 or particles may pile more on one end than an opposing end when the receptacle is positioned in a vertical orientation). For example, a top surface defined by the settled biological cells 330 or particles may be non-perpendicular to the verticaldirection while the receptacle holder 410 or the receptacle 300 is in the vertical orientation as shown in Figure 4D.

[0040] Figure 4E illustrates that, in some embodiments, one or more mechanical impulses are provided to the receptacle 310 or the receptacle holder 410. In some cases, the one or more mechanical impulses cause the biological cells 330 or particles to redistribute. In some cases, the redistribution of the biological cells 330 or particles causes a top surface defined by the settled biological cells 330 or particles to be substantially perpendicular to the vertical direction while the receptacle holder 410 or the receptacle 300 is in the vertical orientation as shown in Figure 4E.

[0041] Figure 4F illustrates an aspirator 450 or its tip. In some embodiments, the aspirator 450 aspirates at least a portion of the solution 320 (and any substances not settled down with the biological cells 330 or particles, such as non-cellular substances).

[0042] Figure 4G illustrates that a portion (e.g., a large portion) of the solution 320 is aspirated. A remaining portion 320-2 of the solution 320 with the biological cells 330 is left in the receptacle 300. The aspirated portion 320-1 is also illustrated in the inset of Figure 4G to show that the aspirated portion 320-1 has no or a low concentration of biological cells whereas the remaining portion 320-2 has a high concentration of biological cells.

[0043] Figure 4H illustrates a dispenser 460 or its tip. In some embodiments, the dispenser 460 is distinct from the aspirator 450. In some embodiments, the dispenser 460 dispenses a different solution (e.g., a wash buffer), which is mixed with the remaining portion of the solution 320 to form a mixed solution 470. By providing the different solution, the concentration of any substances not settled down with the biological cells 330 or particles, such as non-cellular substances, decreases.

[0044] In some embodiments, the aspiration of a portion of a solution in the receptacle and dispensing of additional solution are repeated to further decrease the concentration of any substances not settled down with the biological cells 330 or particles, such as non-cellular substances.

[0045] In some embodiments, the dispenser 460 or another dispenser provides one or more reagents.

[0046] Figure 41 illustrates the aspirator 450 or its tip. In some embodiments, the aspirator 450 aspirates at least a portion of the solution 470 (and any substances not settled down with the biological cells 330 or particles, such as non-cellular substances).

[0047] Figure 4J illustrates that a portion (e.g., a large portion) of the solution 370 is aspirated. A remaining portion 470-2 of the solution 470 with the biological cells 330 is left in the receptacle 300. The aspirated portion 470-1 is also illustrated in the inset of Figure 4J to show that the aspirated portion 470-1 has no or a low concentration of biological cells whereas the remaining portion 470-2 has a high concentration of biological cells.

[0048] Figure 4K illustrates the dispenser 460 or its tip. In some embodiments, the dispenser 460 dispenses a solution (e.g., an incubation buffer), which is mixed with the remaining portion of the solution 470 to form a mixed solution 480.

[0049] Figure 4L illustrates the aspirator 450 or its tip. In some embodiments, the aspirator 450 aspirates at least a portion of the solution 480.

[0050] Figure 4M illustrates that a portion (e.g., a large portion or the entirety) of the solution 480 is aspirated. The aspirated portion 480-1 is also illustrated in the inset of Figure 4M to show that the aspirated portion 480-1 has a high concentration of biological cells 330 whereas the remaining portion 470-2 has no or a low concentration of biological cells.

[0051] Although the operations in Figures 4A-4M are illustrated with a tube, corresponding operations may be performed using a plate (e.g., the plate 100 shown in Figures 1 and 2). For brevity, such details are not repeated herein. The use of a plate having multiple channels allows concurrent separation operations for multiple samples or solutions.

[0052] Figure 5 illustrates experimental results obtained by using the methods described herein.

[0053] Figure 5 illustrates, on the left side, numbers of thawed peripheral blood mononuclear cells (PBMC) washed by a centrifuge-based method and a centrifuge-free washing (CFW) method. For this comparison, a block of cryopreserved PBMC was thawed and split into two separate samples. One sample was washed using centrifugation (e.g., formation of the sediments by centrifugation, followed by aspiration of the solution and addition of a new buffer). The other sample was washed using the methods described herein, without using centrifugation. The results show that the number of cells retained after a washing step is comparable between centrifuge-based washing and centrifuge-less washing.

[0054] Figure 5 also illustrates, on the right side, the viability of thawed PBMC washed by the centrifuge-based method and the centrifuge-free washing. The results show that the viability of thawed PBMC after a washing step is comparable between centrifuge-based washing and centrifuge-less washing.

[0055] In light of these principles and examples, we now turn to certain embodiments.

[0056] In accordance with some embodiments, a method includes maintaining a receptacle with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time (e.g., Figures 4B and 4C); and subsequent to maintaining the receptacle in the first orientation for the first period of time, placing the receptacle in a second orientation distinct from the first orientation (e.g., Figure 4D). The second orientation has a second angle distinct from the first angle with respect to the vertical axis. The method also includes aspirating the first liquid in the receptacle while leaving the cells in the receptacle (e.g., Figures 4F-4G); dispensing a second liquid into the receptacle (e.g., Figure 4H); and aspirating the second liquid in the receptacle while leaving the cells in the receptacle (e.g., Figures 4I-4J).

[0057] In some embodiments, the first angle is a non-zero angle.

[0058] In some embodiments, the second angle is less than the first angle.

[0059] In some embodiments, the second angle is zero.

[0060] In some embodiments, the method includes, prior to maintaining the receptacle with the first liquid in the first orientation, placing in the first orientation the receptacle that had been in a third orientation having a third angle distinct from the first angle with respect to the vertical axis (e.g., Figure 4A).

[0061] In some embodiments, the second angle and the third angle are substantially identical.

[0062] In some embodiments, the method includes, prior to maintaining the receptacle with the first liquid in the first orientation: placing cryopreserved cells into the receptacle; and heating the cryopreserved cells.

[0063] In some embodiments, the method includes, prior to maintaining the receptacle with the first liquid in the first orientation, dispensing the first liquid containing cells that had been cryopreserved into the receptacle.

[0064] In some embodiments, the method includes, prior to maintaining the receptacle with the first liquid in the first orientation: placing cryopreserved cells into the receptacle; and dispensing the first liquid into the receptacle.

[0065] In some embodiments, the first liquid includes an incubation buffer.

[0066] In some embodiments, the second liquid includes a wash buffer.

[0067] In some embodiments, the first liquid and the second liquid are substantially identical in composition.

[0068] In some embodiments, the method includes repeating the dispensing of the second liquid and the aspiration of the second liquid.

[0069] In some embodiments, the method includes dispensing a third liquid into the receptacle (e.g., Figure 4K); and aspirating the third liquid in the receptacle with the cells for transporting the cells (e.g., Figures 4L and 4M).

[0070] In some embodiments, the third liquid includes an incubation buffer.

[0071] In some embodiments, the first liquid has a greater volume than the second liquid.

[0072] In accordance with some embodiments, an apparatus includes a receptacle holder (e.g., receptacle holder 410) for holding a receptacle (e.g., receptacle 310); a tilting device (e.g., tilting device 420) coupled with the receptacle holder for placing the receptacle holder in a first orientation at a first time and placing the receptacle holder in a second orientation distinct from the first orientation at a second time distinct from the first time; one or more liquid handling devices (e.g., aspirator 450 and / or dispenser 460); one or more processors (e.g., processor 412); and memory (e.g., memory 414) storing one or more programs for execution by the one or more processors. The one or more programs include instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle in a second orientation distinct from the first orientation; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the cells in the receptacle. The second orientation has a second angle distinct from the first angle with respect to the vertical axis.

[0073] In some embodiments, the one or more liquid handling devices include an aspirator.

[0074] In some embodiments, the one or more liquid handling devices include a dispenser.

[0075] In some embodiments, the one or more liquid handling devices include a pipette for aspirating and dispensing liquid.

[0076] In accordance with some embodiments, a (non-transitory) computer-readable storage medium storing one or more programs for execution by one or more processors in communication with a tilting device and one or more liquid handling devices, the one or more programs including instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle holder in a second orientation distinct from the first orientation; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the cells in the receptacle. The second orientation has a second angle distinct from the first angle with respect to the vertical axis.

[0077] The foregoing description, for purpose of explanation, has been described with reference to specific embodiments. However, the illustrative discussions above are not intended to be exhaustive or to limit the embodiments to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

Claims

What is claimed is:

1. A method for washing cells that had been cryopreserved, comprising: maintaining a receptacle with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; subsequent to maintaining the receptacle in the first orientation for the first period of time, placing the receptacle in a second orientation distinct from the first orientation, the second orientation having a second angle distinct from the first angle with respect to the vertical axis; aspirating the first liquid in the receptacle while leaving the cells in the receptacle; dispensing a second liquid into the receptacle; and aspirating the second liquid in the receptacle while leaving the cells in the receptacle.

2. The method of claim 1, wherein the first angle is a non-zero angle.

3. The method of claim 1 or 2, wherein the second angle is less than the first angle.

4. The method of any of claims 1-3, wherein the second angle is zero.

5. The method of any of claims 1-4, further comprising: prior to maintaining the receptacle with the first liquid in the first orientation, placing in the first orientation the receptacle that had been in a third orientation having a third angle distinct from the first angle with respect to the vertical axis.

6. The method of claim 5, wherein the second angle and the third angle are substantially identical.

7. The method of any of claims 1-6, further comprising: prior to maintaining the receptacle with the first liquid in the first orientation: placing cryopreserved cells into the receptacle; and heating the cryopreserved cells.

8. The method of any of claims 1-6, further comprising: prior to maintaining the receptacle with the first liquid in the first orientation, dispensing the first liquid containing cells that had been cryopreserved into the receptacle.

9. The method of any of claims 1-6, further comprising:prior to maintaining the receptacle with the first liquid in the first orientation: placing cryopreserved cells into the receptacle; and dispensing the first liquid into the receptacle.

10. The method of any of claims 1-9, wherein the first liquid includes an incubation buffer.

11. The method of any of claims 1-10, wherein the second liquid includes a wash buffer.

12. The method of any of claims 1-10, wherein the first liquid and the second liquid are substantially identical in composition.

13. The method of any of claims 1-12, further comprising: repeating the dispensing of the second liquid and the aspiration of the second liquid.

14. The method of any of claims 1-13, further comprising: dispensing a third liquid into the receptacle; and aspirating the third liquid in the receptacle with the cells for transporting the cells.

15. The method of claim 14, wherein: the third liquid includes an incubation buffer.

16. The method of any of claims 1-15, wherein: the first liquid has a greater volume than the second liquid.

17. An apparatus, comprising: a receptacle holder for holding a receptacle; a tilting device coupled with the receptacle holder for placing the receptacle holder in a first orientation at a first time and placing the receptacle holder in a second orientation distinct from the first orientation at a second time distinct from the first time; one or more liquid handling devices; one or more processors; and memory storing one or more programs for execution by the one or more processors, the one or more programs include instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time;causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle in a second orientation distinct from the first orientation, the second orientation having a second angle distinct from the first angle with respect to the vertical axis; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the cells in the receptacle.

18. The apparatus of claim 17, wherein: the one or more liquid handling devices include an aspirator.

19. The apparatus of claim 17 or 18, wherein: the one or more liquid handling devices include a dispenser.

20. The apparatus of any of claims 17-19, wherein: the one or more liquid handling devices include a pipette for aspirating and dispensing liquid.

21. The apparatus of any of claims 17-20, wherein: the one or more programs include instructions for performing the method of any of claims 2-16.

22. A computer-readable storage medium storing one or more programs for execution by one or more processors in communication with a tilting device and one or more liquid handling devices, the one or more programs including instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle holder in a second orientation distinct from the first orientation, the second orientation having a second angle distinct from the first angle with respect to the vertical axis;causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the cells in the receptacle.

23. The computer-readable storage medium of claim 22, wherein: the one or more programs include instructions for performing the method any of claims 2-16.

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