WO2014105581A1

WO2014105581A1 - Apparatus and method for rapid 3d cell culture analysis using paper stacks

Info

Publication number: WO2014105581A1
Application number: PCT/US2013/076259
Authority: WO
Inventors: Noriaki Yamamoto
Original assignee: Konica Minolta Laboratory U.S.A., Inc.
Priority date: 2012-12-26
Filing date: 2013-12-18
Publication date: 2014-07-03

Abstract

In a paper-based 3D cell culture system, improved methods for preparing and handling stacks of paper for cell culture using one of the following configurations: (1) a continuous long sheet of paper zigzag folded into a stack; (2) a continuous long sheet of paper rolled into a roll; (3) multiple individual sheets bound at one edge into a stack; (4) multiple individual sheets adhered to each other at alternating edges using a low-tack adhesive to form a stack; (5) multiple individual sheets interfolded and stacked. Culture zones are formed on the paper by printing a hydrophobic material on the sheet to separate the zones. Zones on multiple layers of the stack are aligned to form a 3D volume. Cells in hydrogel are deposited into the culture zones, and the sheets are stacked into a stack and cultured. Afterwards, the sheets in the stack are separated and analyzed individually.

Description

APPARATUS AND METHOD FOR RAPID 3D CELL CULTURE ANALYSIS USING

PAPER STACKS

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to paper-based three-dimensional (3D) cell culture, and in particular, it relates to methods for stacking multiple sheets of culture paper and for handling the stack for analysis.

Description of Related Art

In vitro 3D cell culture is gaining attention in recent years because it is a good model for tissues and organs in vivo. 3D cell culture is described in, for example, U.S. Pat. Appl. Pub. Nos. 2009/0221022, 2011/0269207, 2011/0105360, etc. However, some current 3D cell culture systems based on hydrogel are not easy to prepare and analyze.

Paper-based 3D cell culture methods have been disclosed, for example, in U.S. Pat. Appl. Pub. No. 2011/0105360. Figs. 1A and IB of that patent application show the fabrication of three-dimensional cellular arrays using plain paper or paper patterned into hydrophilic and hydrophobic regions. Figs. 6, 7 and 15-19 of that application illustrate various embodiments where sheets of paper are stacked for culture, and then separated (de-stacked) so that the cells grown on each individual sheet can be analyzed.

Paper-based 3D cell culture is also described in Derda R, Tang SKY, Laromaine A,

Mosadegh B, Hong E, et al. (2011) Multizone Paper Platform for 3D Cell Cultures, PLoS ONE 6(5): el8940 (doi: 10.1371/journal.pone.0018940) (hereinafter "Derda et al. 2011"). The abstract of that paper describes: "In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other

environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low- throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously ("cells-in-gels-in-paper" or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De- stacking the layers of the 3D culture, by peeling apart the sheets of paper, "sections" all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures."

SUMMARY

The present invention is directed to a method and related products and apparatus to facilitate easy to use, high-throughput 3D cell culture preparation and analysis.

An object of the present invention is to provide a system useful for handling wet paper encountered in paper-based 3D cell culture systems.

Additional features and advantages of the invention will be set forth in the descriptions that follow and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims thereof as well as the appended drawings.

To achieve these and/or other objects, as embodied and broadly described, the present invention provides a method for 3D cell culture, which includes: providing a continuous sheet of paper, the paper having pre-formed folding lines for folding the paper into a zigzag shaped stack containing multiple layers; depositing cells on the sheet of paper; folding the sheet of paper along the folding lines into a zigzag shaped stack containing multiple layers; culturing the cells in the stack; and de- stacking the stack by pulling the continuous sheet with a tractor feeding device of roller feeding device.

In another aspect, the present invention provides a method for 3D cell culture, which includes: providing a continuous sheet of paper; depositing cells on the sheet of paper; rolling the sheet of paper around a core into a roll containing multiple layers forming a stack; culturing the cells in the stack; and de- stacking the roll by pulling the continuous sheet with a tractor feeding device of roller feeding device.

In another aspect, the present invention provides a method for 3D cell culture, which includes: providing a stack of individual sheets of paper, the sheets being bound at one edge into a bound stack; depositing cells on the sheets of paper; culturing the cells in the stack; and de- stacking and analyzing the sheets of paper by turning each individual sheet of the bound stack.

In another aspect, the present invention provides a method for 3D cell culture, which includes: depositing cells on a plurality of individual sheets of paper, each sheet containing a low tack adhesive material may on one side near an edge thereof; stacking the individual sheets of paper into a stack, each sheet in the stack being adhered to a sheet above it at one edge and adhered to a sheet below it at an opposite edge; culturing the cells in the stack; and de-stacking the sheets by pulling each sheet to separate it from a sheet below it.

In another aspect, the present invention provides a method for 3D cell culture, which includes: depositing cells on a plurality of individual sheets of paper; folding each sheet along a folding line near its center to form a top layer and a bottom layer overlapping each other;

stacking the folded sheets of paper into a stack, wherein the folding lines of alternating sheets are located at opposite sides of the stack, and wherein the top layer of each sheet is inserted between top and bottom layers of a sheet above it, and the bottom layer of each sheet is inserted between top and bottom layers of a sheet below it; culturing the cells in the stack; and de-stacking the sheets by pulling each sheet to separate it from a sheet below it.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically illustrates a paper-based 3D cell culture method.

Figure 2 schematically illustrates multiple sheets stacked in a 3D stack for cell culture.

Figures 3A and 3B schematically illustrate a continuous sheet folded into a fan-fold arrangement for 3D cell culture according to a first embodiment of the present invention. Figures 4A and 4B schematically illustrate two examples of continuous paper feeding device useful in various embodiments of the present invention.

Figures 5A and 5B schematically illustrate two examples of a continuous sheet stacked in a roll for 3D cell culture according to a second embodiment of the present invention.

Figures 6A and 6B schematically illustrate multiple sheets of paper bound into a book form for 3D cell culture according to a third embodiment of the present invention.

Figures 7A and 7B schematically illustrate multiple sheets of paper stacked in a fan-fold arrangement form for 3D cell culture according to a fourth embodiment of the present invention.

Figures 8A and 8B schematically illustrate multiple sheets of paper interfolded and stacked in a stack for 3D cell culture according to a fifth embodiment of the present invention.

Figures 9A and 9B schematically illustrate multiple sheets of paper stacked in a stack for 3D cell culture according to a sixth embodiment of the present invention.

Figures 10A to IOC schematically illustrate multiple sheets of paper folded multiple times into a stack for 3D cell culture according to a seventh embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In a 3D cell culture and analysis system using stacked paper, the paper (or other suitable thin medium, such as film) is used as the cell culture scaffold. Paper is a desirable substrate for cell culture because of its high strength, low toxicity, porous and hydrophilic properties. Each sheet of paper includes cell-permeable areas (referred to as culture zones) separated by cell- impermeable areas. The culture zones may form an array on each sheet, such as a 96-zone array. When stacked, the culture zones of multiple sheets are aligned to form a 3D volume. The cells, contained in hydrogel, are introduced in the culture zones on one or more sheets of paper, and can migrate from sheet to sheet in a tight stack.

A 3D cell culture and analysis method using stacked paper (or other suitable thin medium) is described with reference to Fig. 1. First, each sheet of paper is treated, such as by solid wax printing, to form hydrophobic areas that separate the culture zones (step S I).

Typically, the culture zones themselves do not need to be printed because paper is naturally porous and hydrophilic. Then, cells suspended in hydrogel are deposited (spotted) on the paper in the culture zones (step S2). The pattern of cells deposited in the culture zones, e.g. which zones are spotted with what cell samples (or not spotted) on each sheet of paper, etc., depend on the purpose of the experiments being conducted. Multiple such sheets of paper are stacked into a 3D stack (step S3), and the cells are cultured in the 3D structure, e.g. by submerging the stack in a growth medium (step S4). Afterwards, the stack of paper sheets is de-stacked for analysis (step S5). The separated sheets of paper are treated as needed and measures individually, e.g. by imaging each sheet using a scanner or camera (step S6). The result, such as the imaged pattern on each sheet, is analyzed as appropriate (step S7). This process is generally known in the art. Fig. 2 (adopted from Fig. 2 of the above-mentioned Derda et al. 2011 paper) schematically illustrates a 3D cell culture system, showing the sheets in a separate form as well as a stacked form.

Embodiments of the present invention provide improvements to the above described paper-based 3D cell culture system. In particular, various method of preparing and handling of stacked paper and related apparatus are disclosed.

In a first embodiment, schematically shown in Figs. 3A-4B, a continuous long sheet of paper 101 is folded into a zigzag form (also referred to as fan-fold or accordion style fold) as illustrated in Figs. 3A and 3B. The folded paper forms a flat stack without gaps between adjacent layers. Note that in Fig. 3B, as well as in Figs. 5A-8B described later, the gaps between layers or sheets in the stack are much exaggerated for better understanding; the layers or sheets in the various embodiments are in fact in contact with each other without gaps when they are stacked for cell culture. The continuous long sheet of paper 101 is pre-formed with folding lines or perforations (hereinafter, collectively folding lines), and also has culture zones formed thereon. The locations of the folding lines and the culture zones are such that when the continuous sheet is folded at the folding lines into a stack, the corresponding culture zones in the stacked layers are aligned on top of each other. Optionally, the sheets may be pre-printed with marks to indicate the culture zone boundaries as well as labels to indicate which culture zones will be aligned with each other in the stack. For example, a zone index number (e.g. 1 to 96 for a 96- zone system) may be printed next to each zone, so that zones with the same index number will be aligned in the stack. Of course, if the entire handling process is automated, as will be described later, printing of such marks or labels will not be necessary; rather, the handling devices will be programmed to keep track of which culture zones on the continuous sheet are aligned with each other. The continuous paper 101 can be handled using a tractor feeding device (see Fig. 4A), much like the way continuous form paper for computer printing was handled. As shown in Fig. 4A, a tractor feeding device has a roller 201 with pins on its periphery; two columns of sprocket holes 101A are correspondingly formed near the side edges of the continuous paper 101 (see Fig. 3A) for engaging with the pins to accomplish feeding. Alternatively, a system of rollers 202 (see Fig. 4B) may be used to feed the continuous paper 101. In the feeding system using either a tractor feeder 201 or a roller feeder 202, additional analysis devices (such as a scanner, a camera, other types of detector, etc.) may be located at places where the continuous sheet is held flat by the feeding device. Fig. 4B schematically illustrates an example where a camera 301 is located between two sets of rollers 202 for imaging the continuous sheet 101 which is fed by the feeding device.

In use, the continuous paper 101 is spotted with cells and then folded into a stack to form the desired 3D condition. After a desired time period, the folded continuous paper 101 is unfolded, for example by using the feeding device described above, and analyzed layer-by-layer using the analysis devices.

In a second embodiment, schematically shown in Fig. 5 A, a continuous long sheet of paper 102 is rolled into a roll around a core 401, forming a tight stack of layers. The continuous sheet 102 is pre-formed with culture zones 102A, which are located at predetermined locations such that when the continuous sheet is rolled on a round core 401, the respective culture zones are aligned on top of each other, as schematically indicated by the short parallel lines in Fig. 5A. The desired locations of the culture zones 102A on the continuous sheet can be calculated using the diameter of the core 401 and the thickness of the sheet. The continuous sheet is optionally pre-printed with marks to indicate the culture zone boundaries as well as labels to indicate which culture zones will be aligned with each other in the stack, e.g., by printing a zone index number (e.g. 1 to 96 for a 96-zone system) next to each zone. Alternatively, the culture zones 102A may be formed after the roll of the sheet is formed; any suitable technique can be employed to form the roll-shaped paper with the culture zones 102A.

In use, cells are spotted on the continuous sheet 102, which is then rolled into a roll and cultured. For de-stacking, the continuous long sheet 102 can be handled in similar manners as the continuous long sheet 101 in the first embodiment, using similar feeding devices as shown in Figs. 4 A and 4B. In a variation, instead of a round core 401, the continuous long sheet of paper 102 may be rolled around a plate-shaped core 402 as shown in Fig. 5B. This stack can be handled in similar manners as the stack in Fig. 5A.

In a third embodiment, schematically shown in Figs. 6 A and 6B, multiple individual sheets of paper 103 are bound together at one edge to form a bound stack in the form of a book. The sheets are pre-formed with culture zones, where corresponding zones on different sheets are located at the same locations so that they are aligned in the bound stack. The sheets are optionally pre -printed with marks to indicate the culture zone boundaries and a zone index for each zone. Any suitable binding structure 103 A may be used. Top and bottom cover sheets or support sheet may be provided.

The bound stack may be in an open state or a closed state as schematically shown in Figs. 6A and Fig. 6B, respectively, similar to an open and closed book. In Fig. 6A, the angle between the two stacks of sheets in the open book is shown to be near 180 degrees, but the bound sheets may also be open by other angles, such as 90 degrees. The book is kept in an open state when depositing cells onto the sheets and when analyzing the sheets, and in the close state during cell culture. The sheets are turned during spotting and analysis steps. In one example, when depositing cells, the book is kept open, forming two stacks (see Fig. 6A); cells are deposited on the top sheet of one stack, and that sheet is flipped (turned) to the other stack, exposing the next sheet under it; cells are then deposited on the next sheet, etc. An impermeable insert sheet may be used to separate the sheet being deposited with cells and the sheet below it. During the analysis process, an imaging device 302 or other analysis devices may be positioned above one of the stacks of the open book, as schematically shown in Fig. 6A, to image or otherwise analyze each sheet individually as the sheets are turned one by one.

Appropriate handling devices may be used to turn the sheets in the cell depositing or analyzing stage. For example, automatic page turning and imaging system have been developed for scanning books, one example of which is a book scanner announced by Dai Nippon Printing, seen in http://cxxtedevice om/book-scamier-takes-250-pages-per-minute/. Alternatively, a tab may be provided on the edge of each page which can be used for turning pages. The tabs may be used for both of automated and manual page turning.

In an alternative embodiment, the bound sheets may be handled such that the sheet being processed (spotted or imaged) is separated from both stacks and is held in place by the handling device for processing. For example, if the open book is in the state shown in Fig. 6A, the sheet being processed may be held upright.

In another alternative embodiment, cell spotting is performed on individual unbound sheets, and the sheets are then bound into a book form for cell culturing.

In a fourth embodiment, schematically shown in Figs. 7A and 7B, multiple individual sheets of paper 104 are stacked, and the adjacent sheets are adhered to each other at alternating edges using a low-tack adhesive material 104A so that the sheets are releasable and re-adherable, similar to a stack of accordion-style sticky notes. In other words, each sheet is adhered to the sheet above it at one edge and adhered to the sheet below it at the opposite edge. For example, as illustrated in Fig. 7 A, the first sheet 104-1 is adhered to the second sheet 104-2 (the one below the first sheet) at their right edges; the second sheet is adhered to the third sheet (the one below the second sheet) at their left edges, etc. The individual sheets are pre-formed with culture zones. The low tack adhesive material may be pre-formed on each sheet on one side near an edge thereof.

In use, individual sheets 104 are spotted with cells in the culture zones, and the sheets are stacked up sheet by sheet to form the 3D structure. After cell culturing, the stack is de-stacked by separating the sheets 104 one at a time. For convenient operation, the stack may be placed in a dispenser box 501 which has an elongated opening 501 A at the top for the leading edge of the sheets 104 to pass through. As illustrated in Fig. 7A, when the top sheet 104-1 is pulled through the opening 501 A, because of the adhesive material at the right edge between the top sheet 104-1 and the second sheet 104-2, the right edge of the second sheet is pulled through the opening, and then the top sheet 104-1 and the second sheet 104-2 are separated, resulting in the configuration shown in Fig. 7B. The second sheet 104-2 can be pulled now, bringing the left edge of the sheet below it out of the opening 501A. The sheets 104 in the stack can be successively separated in this manner. After they are separated, the sheets are analyzed individually.

In a fifth embodiment, schematically shown in Figs. 8A and 8B, multiple individual sheets of paper 105 are each folded once near a center line to form a top layer and a bottom layer overlapping each other. When stacked, the folding lines of alternating sheets are located at opposite sides of the stack; the top layer of each sheet is inserted between the top and bottom layers of the sheet above it, and the bottom layer of each sheet is inserted between the top and bottom layers of the sheet below it, as shown in Figs. 8 A and 8B. This is commonly referred to as an interfold configuration. Each sheet of paper 105 is pre-formed with a folding line, as well as culture zones at appropriate locations such that when the sheets are folded and stacked in the above described manner, corresponding zones on the individual sheets (including top layer and bottom layer of the same sheet) will be aligned with each other. Optionally, a zone index number may be printed next to each zone to indication which zones will be aligned with each other in the stack. In addition, because of the manner of stacking described above, the top layer and bottom layer of the same sheet are separated by other layers in the stack. Therefore, each half of each sheet is optionally printed with a layer index to indicate the position of that layer in the stack.

In use, cells are spotted on individual sheets in the culture zones, and the sheets are then folded and stacked in the manner described above for cell culture. To de-stack, as shown in Figs. 8A and 8B, the top layer of the top sheet 105-1 is pulled; as the bottom layer of the top sheet separates from the stack, it brings the top layer of the second sheet 105-2 upwards. After the top sheet 105-1 is removed, the top layer of the second sheet 105-2 becomes free, as shown in Fig. 8B, and can be pulled. In this manner, the folded and stacked sheets are successively pulled and separated. A dispenser box similar to box 501 shown in Figs. 7 A and 7B may be used for convenient de- stacking.

The de-stacking process of the fourth and fifth embodiment can be automated, for example, by using a feed mechanism similar to those used in printer feeders, with suitable modifications. Once the leading part of each sheet (which is often more than half of the sheet) of the top layer is detached, as shown in Figs. 7A-8B, the entire sheet can be pulled with the feed mechanism.

In a sixth embodiment, shown in Figs. 9A and 9B, multiple individual sheets of paper 106 are formed into a stack; each sheet of paper is coated with a low tack adhesive material distributed throughout (but it does not have to be contiguous), except in a narrow area 106A which is free of the adhesive material. The non-adhesion area 106A at the edge may be treated with a hydrophobic material. Culture zones are formed on each sheet at the same locations, such that the zones will be aligned in the stack to form a 3D volume. When stacked, the sheets adhere to each other to make the stack more stable. When de-stacking, the non-adhesion areas 106A can be pulled to separate the sheets in the stack. A paper feeding mechanism, similar to these used in printers, may be used to automatically de- stack the sheets. In a seventh embodiment, shown in Figs. 10A to IOC, a large sheet of paper can be folded multiple times to become a stack of layers. For example, when the sheet of paper 107 shown in Fig. 10A is folded once along the line 107 A, it becomes the shape shown in Fig. 10B, now a stack of two layers. When it is folded again along the line 107B (perpendicular to line 107 A), it becomes the shape shown in Fig. IOC, now a stack of four layers. The culture zones can be formed in the unfolded sheet 107 in such an arrangement that when the sheet is folded the desired times, multiple zones will be aligned with each other in the resulting stack to form 3D volume. De-stacking the stack involves unfolding the folded sheet, which involves an operation similar to turning a page in a book. Appropriate machines may be used to handle the de-stacking (page-turning) operation.

In all embodiments described above, the sheet or sheets of paper may be handled manually during cell spotting, stacking, de-stacking and imaging. However, it is desirable that all paper handling steps, including spotting, stacking, de-stacking and imaging, be automated to achieve high throughput operation.

It can be seen that in many embodiments described above, the de-stacking is performed layer-by-layer in a repeated action.

It will be apparent to those skilled in the art that various modification and variations can be made in the paper-based 3D cell culture method and related apparatus of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover modifications and variations that come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method for 3D cell culture, comprising:

providing a continuous sheet of thin medium, the thin medium having pre-formed folding lines for folding the thin medium into a zigzag shaped stack containing multiple layers;

depositing cells on the sheet of thin medium;

folding the sheet of thin medium along the folding lines into a zigzag shaped stack containing multiple layers;

culturing the cells in the stack; and

de-stacking the stack by pulling the continuous sheet of thin medium with a tractor feeding device or roller feeding device.

2. The method of claim 1, wherein the continuous sheet further has a plurality of sprocket holes aligned in a pair of lines perpendicular to the pre-formed folding lines, and wherein the tractor feeding device has a pair of tracking wheels that gear with the sprocket holes to de-stacks the continuous sheet.

3. The method of claim 1, wherein the continuous sheet of thin medium has a plurality of culture zones that are aligned with each other when the sheet is folded into a stack to form at least one 3D culture zone in the multiple layers, and wherein in the depositing step, the cells are deposited into some of the plurality of culture zones.

4. The method of claim 1, wherein the thin medium is paper.

5. The method of claim 1, further comprising imaging the thin medium while de-stacking the stack.

6. A method for 3D cell culture, comprising:

providing a continuous sheet of thin medium;

depositing cells on the sheet of thin medium;

rolling the sheet of thin medium around a core into a roll containing multiple layers forming a stack; culturing the cells in the stack; and

de-stacking the roll by pulling the continuous sheet of thin medium with a tractor feeding device or roller feeding device.

7. The method of claim 6, wherein the continuous sheet of thin medium has a plurality of culture zones that are aligned with each other when the sheet is rolled into a roll to form at least one 3D culture zone in the multiple layers, and wherein in the depositing step, the cells are deposited into some of the plurality of culture zones.

8 The method of claim 6, wherein the thin medium is paper.

9. The method of claim 6, further comprising imaging the thin medium while de-stacking the roll.

10. The method of claim 6, wherein the core has a round shape.

11. The method of claim 6, wherein the core is plate-shaped.

12. A method for 3D cell culture, comprising:

providing a stack of individual sheets of thin medium, the sheets being bound at one edge into a bound stack;

depositing cells on the sheets of thin medium;

culturing the cells in the stack; and

de-stacking the bound stack by turning individual sheets of the bound stack.

13. The method of claim 12, wherein each individual sheet of thin medium has a plurality of culture zones, wherein the culture zones of the sheets are aligned with each other when the sheets are bound into the bound stack to form at least one 3D culture zone, and wherein in the depositing step, the cells are deposited into some of the plurality of culture zones.

14. The method of claim 12, wherein the thin medium is paper.

15. The method of claim 12, further comprising imaging the individual sheets of thin medium while de- stacking the bound stack.

16. A method for 3D cell culture, comprising:

depositing cells on a plurality of individual sheets of thin medium, each sheet having a low tack adhesive material on one side near an edge thereof;

stacking the individual sheets of thin medium into a stack, each sheet in the stack being adhered to a sheet above it at one edge by the low tack adhesive material on the sheet or the sheet above it, and adhered to a sheet below it at an opposite edge by the low tack adhesive material on the sheet or the sheet below it;

culturing the cells in the stack; and

de-stacking the sheets by pulling each sheet to separate it from the sheet below it.

17. The method of claim 16, wherein each individual sheet of thin medium has a plurality of culture zones, wherein the culture zones of the sheets are aligned with each other when the sheets are stacked into the stack to form at least one 3D culture zone, and wherein in the depositing step, the cells are deposited into some of the plurality of culture zones.

18. The method of claim 16, wherein the thin medium is paper.

19. The method of claim 16, further comprising imaging the sheets after de-stacking them.

20. The method of claim 16, wherein the de-stacking step includes placing the stack in a dispenser, the dispenser having an elongated slit through which the individual sheets are pulled out.

21. A method for 3D cell culture, comprising:

depositing cells on a plurality of individual sheets of thin medium;

folding each sheet along a folding line near its center to form a top layer and a bottom layer overlapping each other; stacking the folded sheets of thin medium into a stack, wherein the folding lines of adjacent sheets are located at opposite sides of the stack, and wherein the top layer of each sheet is inserted between top and bottom layers of a sheet above it, and the bottom layer of each sheet is inserted between top and bottom layers of a sheet below it;

culturing the cells in the stack; and

de-stacking the sheets by pulling the top layer of each sheet to separate it from the sheet below it.

22. The method of claim 21, wherein each individual sheet of thin medium has a plurality of culture zones, wherein the culture zones of the sheets are aligned with each other when the sheets are folded and stacked into the stack to form at least one 3D culture zone, and wherein in the depositing step, the cells are deposited into some of the plurality of culture zones.

23. The method of claim 21, wherein the thin medium is paper.

24. The method of claim 21, further comprising unfolding the sheets after de-stacking them, and imaging the unfolded sheets.

25. The method of claim 21, wherein the de- stacking step includes placing the stack in a dispenser, the dispenser having an elongated slit through which the individual sheets are pulled out.

26. A 3D cell culture system comprising a stack formed by a plurality of layers made of a single continuous thin medium, the plurality of layers respectively having culture zones that are aligned with each other to form at least one 3D culture zone in the plurality of layers.

27. The system of claim 26, wherein the thin medium is paper.

28. A 3D cell culture system comprising a single continuous thin medium rolled around a core into a roll, the roll having a plurality of layers forming a stack, the thin medium having culture zones that are aligned with each other in the roll to form at least one 3D culture zone in the plurality of layers.

29. The system of claim 28, wherein the thin medium is paper.

30. A 3D cell culture system comprising a stack formed by a plurality of layers made of separate sheets of thin media, the layers being bound together at one side, the plurality of layers respectively having culture zones that are aligned with each other to form at least one 3D culture zone through the plurality of layers.

31. The system of claim 30, wherein the thin medium is paper.

32. A 3D cell culture system comprising a stack formed by a plurality of layers made of separate sheets of thin media, the plurality of layers respectively having culture zones that are aligned with each other to form at least one 3D culture zone through the plurality of layers, wherein the plurality of layers are stacked on top of each other in a manner that when a top one of the layers is detached from the stack the next top one of the layers is partly detached from the stack.

33. The system of claim 32, wherein the thin medium is paper.

34. A 3D cell culture system comprising a stack formed by a plurality of layers made of separate sheets of thin media, each sheet including an adhesion area having an adhesive material distribute throughout and a non-adhesion area free of the adhesive material, the non-adhesion area being located on one side of the sheet, the plurality of layers respectively having culture zones that are aligned with each other to form at least one 3D culture zone through the plurality of layers.

35. The system of claim 34, wherein the thin medium is paper.

36. A 3D cell culture system comprising a stack formed by a plurality of layers made of a single thin medium folded a first time along a first line and then folded a second time along a second line perpendicular to the first line, the plurality of layers respectively having culture zones that are aligned with each other to form at least one 3D culture zone in the plurality of layers.

37. The system of claim 36, wherein the thin medium is paper.