Methods for the handling of cells, cell clusters, and / or organoids

Abstract

The present disclosure pertains to methods for handling cells and / or cell clusters without the use of centrifugation. In one aspect, described is a method of separating a liquid from a composition comprising the liquid and cell clusters. Therein, the liquid of the composition is passed through a filter disposed within a pipette so as to separate the liquid from the cell clusters.

Description

[0001] METHODS FOR THE HANDLING OF CELLS, CELL CLUSTERS, AND / OR ORGANOIDS

[0002] TECHNICAL FIELD

[0003] The present disclosure generally relates to methods of handling cells. Such methods find application in, amongst other, in cell culture and maintenance, providing methods for the efficient handling (such as passaging) of cells and cell clusters such as organoids. The methods described herein allow for the easier and more effective handling of cells and / or cell clusters without the need for centrifugation to separate the cells and / or cell clusters or debris from a cell culture medium.

[0004] BACKGROUND

[0005] Cell culture has become an indispensable tool in various scientific and medical disciplines, ranging from basic research to pharmaceutical and biotechnological applications. Cells and / or cell clusters require periodic passaging to maintain their health, viability, and proliferative capacity. Conventional cell passaging methods involve centrifugation to separate cells and / or cell clusters from liquid such as growth medium which may subject the cells to mechanical stress, potentially leading to altered cell behavior, reduced viability, or even cell death.

[0006] SUMMARY

[0007] Various methods for handling cells are described.

[0008] In one aspect, described is a method of separating a liquid from a composition comprising the liquid and cell clusters. Therein, the liquid of the composition is passed through a filter disposed within a pipette so as to separate the liquid from the cell clusters.

[0009] In another aspect, the liquid is a cell handling / culturing liquid such as culture medium or passaging medium. In further aspects the cells clusters may be organoids, spheroids, microtissues, or tumoroids.

[0010] In aspects, the filter substantially impedes the passage of the cell clusters when liquid containing the cell clusters passes through the filter. Thus, liquid passed through the filter should contain significantly reduced numbers of cell clusters as compared to the liquid before passing through the filter. In particular aspects, the filter has a pass-through size that is smaller than the size of the average cell cluster suspended in the liquid. In some aspects the filter is a resin. In further aspects, the filter has a particle size of 20pm or 50pm. In aspects the pipette is a pipette tip or micropipette tip and may or may not be disposable. In particular aspects the pipette tip or micropipette tip is designed for use with a second pipette tip in a tip-on-tip configuration. In certain aspects the second pipette tip comprises one or more second filters. In other aspects the first pipette tip comprises no filter, and the second pipette tip comprises one or more second filters.

[0011] In some aspects, included in the method is inserting the tip of the pipette just under the surface of the liquid and keeping the position of the pipette in the same position relative to the surface as the liquids is drawn into the pipette. In particular aspects, the liquid is drawn into the pipette or passed through the filter at a rate of 200pl / s or less or 50pl / s or less. In aspects the drawing of the liquid into the pipette is performed by an automated liquid handling device.

[0012] In additional aspects, the method includes suspending the cell clusters in the liquid in a container and aspirating the liquid into the pipette so as to pass the liquid through the filter. In aspects, the method includes removing the cell clusters from a three-dimensional cell growth matrix and, after aspirating, resuspending the cell clusters in a new three three- dimensional cell growth matrix. In embodiments, the three-dimensional cell growth matrix may include a temperature sensitive three-dimensional cell growth matrix.

[0013] In some aspects the container containing the cell clusters suspended in the liquid is a cell culture dish, such as a multi-well plate.

[0014] In any of the methods described herein, centrifugation of the cell clusters need not be performed or is not performed as part of passaging the cells and / or cell clusters.

[0015] Described herein are methods of removing debris from cells where the debris is larger than the cells. The method comprises passing the cells and liquid through a filter disposed within a pipette so as to remove the larger debris that was present in the liquid.

[0016] In another aspect, the liquid is a cell handling / culturing liquid such as culture medium or passaging medium. In further aspects the cells clusters may be organoids, spheroids, microtissues, or tumoroids.

[0017] In aspects, the filter substantially impedes the passage of the larger when liquid containing the cells passes through the filter. Thus, liquid passed through the filter should contain significantly reduced larger debris as compared to the liquid before passing through the filter. In particular aspects, the filter has a pass-through size that is larger than the size of the average cell suspended in the liquid but smaller than the average size of the larger debris. In some aspects the filter is a resin. In further aspects, the filter has a particle size of 20pm or 50pm. In aspects the pipette is a pipette tip or micropipette tip and may or may not be disposable. In particular aspects the pipette tip or micropipette tip is designed for use with a second pipette tip in a tip-on-tip configuration. In certain aspects the second pipette tip comprises one or more second filters. In other aspects the first pipette tip comprises no filter, and the second pipette tip comprises one or more second filters.

[0018] In some aspects, included in the method is inserting the tip of the pipette just under the surface of the liquid and keeping the position of the pipette in about the same position relative to the surface as the liquids is drawn into the pipette. In particular aspects, the liquid is drawn into the pipette or passed through the filter at a rate of 200pl / s or less or 50pl / s or less. In aspects the drawing of the liquid into the pipette is performed by an automated liquid handling device. In yet another aspect a first pipette without a filter is used to aspirate cells and liquid, and the aspirated contents of the first pipette passed thought a second pipette comprising one or more filters.

[0019] In additional aspects, the method includes aspirating a composition having cells and larger debris into a pipette. A second pipette having a filter disposed there is affixed to the pipette and the liquid is dispensed through the filter so as to retain the larger debris.

[0020] It should be understood that language used in the present disclosure has been principally selected for readability and instructional purposes, and not to limit the scope of the subject matter disclosed herein.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 provides a schematic representation of prior methods of passaging cells or cell clusters.

[0023] Figure 2 provides an example of a method of passaging cells without centrifugation as disclosed herein.

[0024] Figure 3 provides an alternate example of a method of passaging cells without centrifugation as disclosed herein.

[0025] Figures 4A through 4H. Images of organoids passaged using a pipette with a 20um particle size filter can be seen in FIGS 4A-4D. Images of organoids passaged using a pipette with a 50um particle size filter can be seen in FIGS 4E-4H.

[0026] Figure 5 shows images of a single cell suspension obtained after passage through a filter. The left panel shows the single cells obtained after passage through the filter. The right panel shows those same cells after seven days in culture. Figure 6 is an image of organoids left behind in a well when the media in which they were suspended was substantially removed using a pipette having a filter disposed therein.

[0027] DETAILED DESCRIPTION

[0028] The present application addresses the limitations of conventional cell passaging methods by introducing a novel approach that allows the avoidance of centrifugation. This gentle and efficient process ensures the maintenance of cell integrity, viability, and desired phenotypic characteristics.

[0029] The methods described herein have several advantages of prior methods:

[0030] Gentle separation of cells and / or cell clusters from liquid: The lack of centrifugation in the described methods ensure a gentle handling of cells, minimizing stress and potential damage.

[0031] Efficiency: The process is highly efficient, allowing for the rapid and reliable transfer of cells without the need for a separate centrifugation step.

[0032] Versatility: The invention is applicable to various cell types including single cells and cell clusters and can be adapted to different culture conditions.

[0033] Maintained Cell Phenotype: By avoiding harsh centripetal forces, the described methods help maintain the desired phenotype and functionality of the cultured cells.

[0034] Reduced Contamination Risk: The closed-system approach reduces the risk of contamination associated with additional handling (z.e. transfer in and out of additional containers and the centrifuge) during conventional passaging methods.

[0035] In conclusion, the methods described herein offer a significant advancement in the field of cell culture, providing a gentle, efficient, and versatile approach to maintaining and propagating cells and or cell clusters.

[0036] The prior methods for passaging cells are generally set forth in FIG. 1. Cell / clusters are enzymatically and / or mechanically dissociated from a culture dish if required. The cells are placed in a tube and centrifuged to form a pellet that allows the culture liquid to be removed. The cell / clusters are then resuspended and seeded for culture.

[0037] In one aspect, described is a method of separating a liquid from a composition comprising the liquid and cell clusters. Therein, the liquid of the composition is passed through a filter disposed within a pipette so as to separate the liquid from the cell clusters. An example of such a method is presented in FIG. 2. Cell / clusters are enzymatically and / or mechanically dissociated from a culture dish if required. The liquid containing the cells / clusters is aspirated into the pipette leaving the cells / clusters behind. The cell / clusters are then resuspended and seeded for culture.

[0038] The liquid can be any liquid used as part of the cell culturing process including, but not limited to culture media, phosphate-buffered saline, passaging media such those containing and / or chelators. Examples of enzymes include trypsin, collagenase, dispase, elatase, proteas XIV, hyaluronidase. Examples of chelators included EDTA, EGTA, BATPA, NTA, and chelating resins.

[0039] In aspects the cells may be any kind of cells including primary cells, stem cells, or cell lines. Cell clusters may be organoids, spheroids, acini, cordons, microtissues, or tumoroids. In addition, the cell clusters may comprise a single cell type (homotypic) or may comprise multiple cell types (heterotypic).

[0040] In aspects, the filter substantially impedes the passage of the cell clusters when liquid containing the cell clusters passes through the filter. Substantially impedes, as used herein, occurs when a liquid passed through a filter contains significantly reduced numbers or amount of a substance suspended in the liquid as compared to the liquid before passing through the filter. Examples of substances which can be filtered / reduced in this way include, but are not limited to cells, cell clusters, and debris that is larger in average size than cells and / or cell clusters. In particular aspects, the filter has a pass-through size that is smaller than the size of the average cell cluster suspended in the liquid, e.g., cell clusters do not pass freely through the filter. In other aspects, the filter may have a pass-through size that is larger than cells or cell clusters but smaller than larger debris, e.g., cells and / or cell clusters will pass freely through the filter while larger debris will not. Examples of filters useful in the methods described herein include, but are not limited to, depth filters, surface filters, screen filters, mesh filters, adsorption filters, resin filters, and membrane filters. In some aspects the filter is a resin.

[0041] In additional aspects, the filter has an internal depth along the long axis of the pipette through which liquid may be drawn through when aspirated into the pipette. The filter depth may of a depth sufficient to substantially impedes passage of a substance that is to be substantially reduced in the liquid. In aspects, the filter depth may be 20, 19, 18, 17, 16, 15, 14, 13 12, 11, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4., 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1mm. In particular aspects the filter has a filter depth of 3.5mm. In other aspects the filter has a filter depth of 3.6mm. The filter may have a filter depth that is selected to work with particular tissue / cells of interest. In other aspects, the filter substantially impedes passage throughout the internal depth of the filter, not just at the surface thereof.

[0042] In further aspects, the filter has a particle size of 500pm. In aspects the filter has a particle size of 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1pm. In particular aspects the filter has a particle size of 20pm. In other aspects the filter has a particle size of 50pm. The filter may have a particle size that is selected to work with particular tissue / cells of interest. In embodiments, the pipettes may be pipettes available from DPX Technologies (dpxtechnologies.com).

[0043] In aspects the pipette is a pipette tip or micropipette tip. The pipette may be configured for attachment to a liquid handling device and / or other pipettes. Examples of liquid handlers include robotic liquid handlers, microfluidic devices, micropipettes, pipetting robots, burettes, syringes and the like. In particular aspects the pipette is designed for use / configure to be attached to a second pipette in a pipette-on-pipette configuration. In such configurations, liquid from one pipette is dispensed directly into the lumen of the second pipette. Where the pipette is a pipette tip, this can be a tip-on-tip configuration. In certain aspects the second pipette comprises one or more additional filters. In a particular embodiments, the second pipette comprises one or two filters.

[0044] In some aspects, included in the method is inserting the tip of the pipette just under the surface of the liquid and keeping the position of the pipette in the same position relative to the surface as the liquids is drawn into the pipette. This methodology eliminates or at least reduces issues that may arise from overflow. In other aspects where overflow is not of concern, the tip of the pipette may be inserted further below the surface of the liquid, in the vicinity of the cells and / or cell clusters. In particular aspects, the liquid is drawn into the pipette or passed through the filter at a rate of 500pl / s or less. In aspects the liquid is drawn into the pipette or passed through the filter at a rate of 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or Ipl / s. In a particular aspect, the liquid is drawn into the pipette or passed through the filter at a rate of 200pl / s or less. In a further particular aspect, the liquid is drawn into the pipette or passed through the filter at a rate of 50pl / s or less. In aspects the liquid is drawn / aspirated into the pipette through the use of a liquid handler. Examples of liquid handlers include robotic liquid handlers, microfluidic devices, micropipettes, pipetting robots, burettes, syringes and the like. Utilization of robotic or other such automated liquid handlers may include liquid level sensors, enabling the level of the liquid to be determined, and for pipette tip placement to be carried out automatically with high precision based on the determined liquid level. In some aspects, the liquid level sensing may be achieved by using, for example, a conductive pipette tip, allowing for capacitive liquid level sensing.

[0045] In additional aspects, the method includes suspending cells and / or cell clusters in the liquid in a container and aspirating the liquid into the pipette so as to pass the liquid through the filter. In some aspects, the method includes removing the cell clusters from a three- dimensional cell growth matrix prior to aspirating with the pipette. After the liquid is removed, the cells and / or cell clusters may be resuspended in fresh cell culture media or in a new three three-dimensional cell growth matrix. In embodiments, the three-dimensional cell growth matrix is a temperature sensitive three-dimensional cell growth matrix. Specific examples of three-dimensional cell growth matrices include Matrigel® and Cultrex®.

[0046] In some aspects the container containing the cell clusters suspended in the liquid is a cell culture dish. Examples of culture dishes include single well plates, multi- well plates, flasks, roller bottles, and hanging drop plates.

[0047] In any of the methods described herein, centrifugation of the cell clusters need not be performed or is not performed as part of passaging the cells and / or cell clusters.

[0048] Also described herein are methods of removing debris from cells where the debris is larger than the cells. The method comprises passing the cells and liquid through a filter disposed within a pipette so as to remove the larger debris that was present in the liquid.

[0049] In additional aspects, the method includes aspirating a composition having cells and larger debris into a pipette. A second pipette having a filter disposed there is affixed to the pipette and the liquid is dispensed through the filter so as to retain the larger debris. An example of such a method is presented in FIG. 3. Cell / clusters are enzymatically and / or mechanically dissociated from a culture dish if required. The liquid containing the cells / clusters is aspirated into the pipette. A second pipette containing a filter is affixed to the first pipette and the liquid containing cells and / or cells clusters is passed through the filter. The cell / clusters are then seeded for culture.

[0050] EXAMPLES

[0051] The present invention is further described in the following examples, which are offered by way of illustration and are not intended to limit the invention in any manner.

[0052] Example 1: Removal of cell culture media from suspended organoid fragments.

[0053] Organoids were cultured in domes of Matrigel® set in wells of a multi-well plate. To passage the organoids, the following method was performed. Cell culture media was removed from the well with dome. 500ul cold cell recovery solution (CRS) was then added to the well. The CRS was spiraled and the plate tilted and CRS squirted at the edges and in the middle of the dome. 500ul of CRS+cells were collected in a Greiner 96 deep well block plate. A microscope was used to determine the # of cells / organoids that remained int the well. The CRS steps were then repeated to ensure that all cells are removed and placed in the deep well block. A DPX tip (20um hydrophobic & 50um) was used to remove media (900ul) from wells in deep well block. The tip was inserted under liquid surface, CRS aspirated slowly, following liquid level with the tip. After aspiration, 2 drops were pushed back into an empty well — to check if there were organoids stuck at on the bottom surface of the filter. After the push back, all of the aspirate was dispensed into another well - and checked if there were organoids that passed through the filter. 800ul of Cold Base media (DMEM F12 + GlutaMax +P / S) was added to the remaining organoid and mix gently lx. Wait ~3min. The same DPX tip was used to aspirate as much media as possible, leaving about 50ul behind. The 50ul cell suspension was then mixed using a 200ul regular pipette tip ~20x to fragment organoids. 200ul of Matrigel was then added and mixed gently. The mixture was then plated by dispensing 40ul per dome per well. Complete media + RI (Rock Inhibitor) was then added to the wells.

[0054] Analysis of the push back revealed that nearly zero cell organoids were stuck at on the bottom surface of the filter. Further no organoids passed through the filter. The replated organoid fragments were cultured for seven days. Images of organoids passaged using a pipette with a 20um particle size filter can be seen in FIGS 4A-4D. Images of organoids passaged using a pipette with a 50um particle size filter can be seen in FIGS 4E-4H.

[0055] Example 2: Removal of cell culture media from single cell suspension.

[0056] Organoids were cultured in domes of Matrigel® set in wells of a multi-well plate. To passage the organoids, the following method was performed. Cell culture media was removed from the well with dome. 500ul cold cell recovery solution (CRS) was then added to the well. The CRS was aspirated and the plate tilted and CRS squirted at the edges and in the middle of the dome. 500ul of CRS+cells were collected in 15mL falcon tube. The tube was then centrifuged and the CRS is aspirated from the pellet. The cells were then resuspended in about 200ul of DMEM. A 200ul pipette tip was then used to aspirate about lOOul of the suspension. The tip containing the suspension was then plugged into a second tip (tip-on-tip) containing a filter having a 20um particle size. The suspension was dispensed through the filter into 2ml Eppendorf tubes. Matrigel® was then added to make up about 80% of the volume. The mixture was then plated by dispensing 40ul per dome per well. Complete media + RI (Rock Inhibitor) was then added to the wells.

[0057] The results are presented in Figure 5. The left panel shows the single cells obtained after passage through the filter. The right panel shows those same cells after seven days in culture.

[0058] Example 3: Using filter tips on an automated liquid handler for organoid passaging.

[0059] Organoids were obtained as in Example 1. Suspensions of organoids in cell culture media were added to the wells of a 1ml deep well plate (800uL). The plate containing the suspensions was then loaded into a Hamilton liquid handler. Regular tips were loaded and the contents of the wells aspirated with liquid detection at a 2mm submerge depth and liquid following to remove 500uL (speed 50uL / s). The aspirate was dispensed to waste and the tips discarded. DPX lOOOul tips were loaded and used to remove 300uL at a 5mm fixed height and a speed of 50uL / s. The remaining contents of the wells were observed under a microscope. An example image of a well is presented as Figure 6. As can be seen there, the method efficiently leaves behind organoids while substantially removing the liquid in which they were suspended.

[0060] In addition, organoids were obtained as in Example 1. Suspensions of organoids in cell culture media (1ml) were added to the wells of a plate. The plate containing the suspensions was then loaded into a liquid handler. Regular tips were loaded and the contents of the wells aspirated without liquid detection at a 30mm above the bottom of the plate and liquid following to remove 500uL. The aspirate was dispensed to waste and the tips discarded. DPX lOOOul tips were loaded and used to remove 400uL at a 15mm height above the bottom of the plate and liquid following. The remaining contents of the wells were observed under a microscope.

[0061] While this invention has been described in certain embodiments, the present invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.

Claims

What Is Claimed:

1. A method of separating liquid from a composition comprising the liquid and cell clusters, the method comprising: passing the liquid of the composition through a filter disposed within a pipette so as to separate the liquid from the clusters.

2. The method according to claim 1, wherein the liquid is a cell culture medium.

3. The method according to claim 1, wherein the cell clusters are organoids.

4. The method according to claim 1, wherein the filter substantially impedes the passage of the cell clusters through the filter.

5. The method according to claim 1, wherein the pipette is a pipette tip for use with a liquid handler.

6. The method according to claim 5, wherein the pipette tip is for use with a micropipette.

7. The method according to claim 5, wherein the pipette tip is a disposable pipette tip.

8. The method according to claim 5, wherein the pipette tip is designed for use with a second pipette tip in a tip-on-tip configuration.

9. The method according to claim 8, where the second pipette tip has one or more second filters.

10. The method according to claim 1, wherein the filter has a pass-through size smaller than an average cell cluster size.

11. The method according to claim 1, wherein the filter is a resin.

12. The method according to claim 1, wherein the filter has a particle size of 20pm or13. The method according to claim 1, wherein the liquid is passed through the filter at rate of 200pl / s or less.

14. The method according to claim 13, wherein the liquid is passed through the filter at rate of 50pl / s or less.

15. The method according to claim 1, wherein passing the liquid of the composition through the filter comprises inserting a tip of the pipette just under a surface of the composition and following a position of the tip relative to the surface as the composition is drawn into the pipette and passed through the filter.

16. The method according to claim 1, wherein the method is performed by an automated liquid handling device.

17. The method according to claim 1, wherein the method comprises: suspending the cell clusters in the liquid in a container so as to create the composition; and aspirating the liquid into the pipette so as to pass the liquid of the composition through the filter so as to substantially remove the liquid from the cell clusters.

18. The method according to claim 17, further comprising removing the cell clusters from a three-dimensional cell growth matrix and, after aspirating, resuspending the cell clusters in new three-dimensional cell growth matrix.

19. The method according to claim 18, wherein the three-dimensional cell growth matrix is a temperature sensitive three-dimensional matrix that supports cell cluster growth.

20. The method according to claim 17, wherein the container is a cell culture dish.

21. The method according to claim 20, wherein the cell culture dish is a multi- well plate.

22. The method according to claim 17, where the method does not comprise centrifuging the cell clusters.

23. The method according to claim 17, wherein the method is performed by an automated cell culture device.

24. A method of removing cells from larger debris in a composition comprising a liquid, cells, and larger debris, the method comprising: passing the cells and liquid of the composition through a filter disposed within a pipette so as to remove the larger debris from the composition.

25. The method according to claim 24, wherein the liquid is a cell culture medium.

26. The method according to claim 24, wherein the filter substantially impedes the passage of the larger debris through the filter.

27. The method according to claim 24, wherein the pipette is a pipette tip for use with a liquid handler.

28. The method according to claim 27, wherein the pipette tip is for use with a micropipette.

29. The method according to claim 27, wherein the pipette tip is a disposable pipette tip.

30. The method according to claim 27, wherein the pipette tip is designed for use with a second pipette tip in a tip-on-tip configuration.

31. The method according to claim 30, where the second pipette tip has one or more second filters.

32. The method according to claim 24, wherein the filter has a pass-through size smaller than the larger debris size.

33. The method according to claim 24, wherein the filter is a resin.

34. The method according to claim 24, wherein the filter has a particle size is 20pm or 50pm.

35. The method according to claim 24, wherein the liquid is passed through the filter at rate of 200pl / s or less.

36. The method according to claim 25, wherein the liquid is passed through the filter at rate of 50pl / s or less.

37. The method according to claim 24, wherein the method is performed by an automated cell culture device.

38. The method according to claim 24, wherein the method comprises: aspirating the composition into the pipette; affixing to the pipette to a second pipette having the filter disposed therein; dispensing the composition through the filter so as to remove the larger debris from the composition.

39. The method according to claim 38, wherein the method is performed by an automated cell culture device.

Images