WO2015101702A1

WO2015101702A1 - Sample holder, cell culture system and mold

Info

Publication number: WO2015101702A1
Application number: PCT/FI2014/050944
Authority: WO
Inventors: Susanna Narkilahti; Laura YLÄ-OUTINEN; Teemu Ihalainen; Tiina JOKI; Joose KREUTZER; Soile NYMARK
Original assignee: Tampereen Yliopisto; Tty-Säätiö
Priority date: 2013-12-30
Filing date: 2014-12-03
Publication date: 2015-07-09

Abstract

The present invention provides a novel sample holder (100) for holding at least one three- dimensional biometric sample for imaging thereof with high resolution optical microscopy. The novel sample holder includes a body portion (110), which has a top surface (111) and bottom surface (112). The sample holder also includes a sample recess (120), which is formed into the body portion for receiving the sample. The sample recess has a top opening (121), which is formed into the top surface (111) of the body portion and which extends over a first area (A1) along the top surface of body portion. The sample recess also has a bottom opening (122), which is formed into the bottom surface of the body portion and which extends over a second area (A2) along said bottom surface of the body portion. The second area is larger than the first area. The sample recess further has a lateral enclosing surface (123), which connects the openings and defines a sample volume (V) between the openings for the three-dimensional sample.

Description

SAMPLE HOLDER, CELL CULTURE SYSTEM AND MOLD

FIELD OF THE INVENTION

The present invention relates to laboratory equipment. In particular, the invention relates to devices for holding three-dimensional (3D) cell or tissue cultures or other tissue engi- neering (TE) related samples. More specifically, the invention relates to a sample holder, cell culture system and mold according to the preamble portions of claims 1, 14 and 18, respectively.

BACKGROUND ART

Laboratory studies performed on biological samples, such as cells, tissue samples, TE scaffolds etc., are designed to imitate real life interaction between organisms and particular substances under investigation. Traditionally experiments have been carried out on cells that were cultivated in an essentially two-dimensional form, i.e. flat samples. It has, however, become increasingly popular to cultivate three-dimensional samples to be experimented with. The reason is that cell cultures extending in three dimensions are able to mimic tissue part/tissue/organ like conditions in more realistic manner.

There exist several sample holders suitable for cultivating 3D samples. US 20070172944 discloses a cell culture system including a cell culture tray and a sample holder that rests on the cell culture tray. The cell culture tray includes a substantially flat surface with more than one well configured to contain a liquid volume and an outer wall surrounding the tray to define a reservoir configured to fluidly interconnect the volume of the more than one well. The top surface of the sample holder of US 20070172944 is as large as its bottom surface.

EP 0590 513 discloses an apparatus for growing tissue cultures in vitro including a sample holder. The top surface of the sample holder of EP 0 590 513 is as large as or larger than the bottom surface.

For imaging purposes the shape of the sample holders of prior art are not optimal. When imaged from the bottom, the sample can be imaged only partially due to the distortions of the light path caused by the vertical walls of the sample recess. Furthermore, unintentional loss of the material from the sample holder may occur.

SUMMARY

The following presents a simplified summary in order to provide a basic understanding of some aspects of various invention embodiments. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary merely presents some concepts of the invention in a simplified form as a prelude to a more detailed description of exemplifying embodiments of the invention. The present invention concerns a sample holder for holding at least one three- dimensional biometric sample for culturing and imaging, preferably by using high resolution optical microscopy thereof. The sample holder includes a body portion, which has a top surface and bottom surface. The sample holder also includes a sample recess, which is formed into the body portion for receiving the sample. The sample recess has a top opening, which is formed into the top surface of the body portion and which extends over a first area along the top surface of body portion. The sample recess also has a bottom opening, which is formed into the bottom surface of the body portion and which extends over a second area along said bottom surface of the body portion. The second area is larger than the first area. The sample recess further has a lateral enclosing surface, which connects the openings and defines a sample volume between the openings for the three- dimensional sample. The sample holder further comprises an imaging surface arranged to close the bottom opening.

A sample holder according to an exemplifying embodiment of the invention constitutes a cell culture system including a cell culture tray and a body portion of the kind described above.

The present invention concerns also a mold having a form suitable for manufacturing, by mold casting, a sample holder according to the invention.

Due to the shape of the sample holder of the present invention, the sample is held in the holder in a stable way because the enclosing surface flares downward thus lowering the center of gravity of the sample and protecting the sample for fluid shear. This is advantageous when experiments are conducted with materials such as hydrogels (i.e. sample is cells and supporting substrate) that adhere poorly to the different materilas and have a tendency to unintentionally exit the sample recess. This is especially important when there is fluid flow within or in close proximity of the sample recess. The stable attachment to culturing platforms and the sample holder is essential also for applications including imaging and electrophysiological measurements.

The shape of the sample recess of the present invention also enables high resolution microscopy because the angle between the bottom surface of the body portion and the en- closing surface is flanked for minimal optical distortion.

BRIEF DESCRIPTION OF DRAWINGS

In the following, exemplary embodiments of the invention are described in greater detail with reference to the accompanying drawings in which:

Fig. 1 presents an isometric view of a sample holder according to one embodiment fea- turing one sample recess,

Fig. 2 presents side cross- sectional view of the sample holder of Fig. 1,

Fig. 3 presents an isometric view of a modification of the sample holder of Fig. 1 featuring four sample recesses,

Fig. 4 presents an isometric view of a sample holder according to another embodiment featuring one sample recess,

Fig. 5 presents side cross- sectional view of the sample holder of Fig. 4,

Fig. 6 presents a top elevation view of the sample holder of Fig. 4,

Fig. 7 presents an isometric view of a modification of the sample holder of Fig. 4 featuring four sample recesses, Fig. 8 presents an isometric view of a sample holder according to yet another embodiment featuring four sample recesses, Fig. 9 presents side cross- sectional view of the sample holder of Fig. 8, and

Fig. 10 presents an isometric view of a modification of the sample holder of Fig. 8 featuring four sample recesses.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Figs. 1 to 3 show a sample holder 100 for cultivating cells, which may be examined within the sample holder on an imaging surface 200. The sample holder 100 may be an insert or a similar device, which may be inserted into a receptive frame for holding studied samples. The sample holder includes a body portion 110, the bottom surface 112 of which is designed to make contact with an imaging surface 200. Alternatively, the imag- ing surface 200 may be an integral part of the body portion 110. The imaging surface 200 may be formed as part of the sample holder 100 by single or multicomponent molding or by means of adhesives, for example. The imaging surface 200 itself may be an optical microscopy suitable coverslip or similar optical substrate used for imaging biometric samples. The body portion 110 may be formed of various suitable materials and shapes. In Figs. 1 to 7 the body portions are prismatic, but also other shapes such as conical or cylindrical (cf. Figs. 8 to 10) are suitable.

In one embodiment, the body portion is formed of a substantially rigid, water-insoluble, fluid- impervious, typically thermoplastic material substantially chemically non-reactive with the fluids to be employed in the assays to be carried out with the sample holder. The term "substantially rigid" as used herein is intended to mean that the material will resist deformation or warping under a light mechanical and/or thermal load, which deformation would prevent maintenance of the substantially planar surface, although the material may be somewhat elastic. Suitable materials include, for example, polystyrene or polyvinyl chloride with or without copolymers, polyethylenes, polystyrenes, polystyrene- acrylonitrile, polypropylene, polyvinylidine chloride, silicone eleastomers and similar materials. Polystyrene is a material that can be used as it is the common polymer used for cell culture vessels, inasmuch as it is characterized by very low, non-specific protein binding, making it suitable for use with biometric samples, such as, for example, tissues, cells, blood, viruses and bacteria. Glass is also a suitable material, being used routinely in cell culture vessels and can be washed and sterilized after each use. In an illustrative ex- ample, the body portion 110 is made from silicone elastomer, such as PDMS, whereby the sample holder 100 may be attached to and detached from an objective, cover glass, coverslips or cell culture plastics etc. without the use of any adhesive. The body portion 110 features a top surface 111 at a distance from the bottom surface 112, whereby a block for housing a sample recess 120 is defined in between.

According to another embodiment (Figs. 4 to 7) the body portion 110 also features an additional collar 110a protruding upwards from the top surface 111 for providing a medium chamber for e.g. cell culturing medium and/or molecules under investigation. The base 110b of the body portion 110 is similar to that as described in connection with Figs. 1 to 3. The volume defined by the collar 110a, i.e. the medium chamber, is particularly for containing cell culturing medium or other solutions allowing easier sample observation in physiological conditions. The body portion 110 therefore includes the base 110b and preferably the collar 110a for providing a medium chamber.

The sample recess 120 formed into the body portion 110 is shaped to receive and stably hold the three-dimensional biometric sample. The sample recess 120 has a top opening 121, which is formed into the top surface 111 of the body portion 110. The top opening 121 extends over a first area Al along the top surface 111, i.e. the inlet opening of the sample recess 120 defines an area Al in the top surface 111 of the body portion 110. The sample recess 120 has also a bottom opening 122, which is formed into the bottom sur- face 112 of the body portion 110. The bottom opening 122 extends over a second area A2 along said bottom surface 112. According to an embodiment the openings 121, 122 are coaxial and preferably aligned to be parallel with the intended imaging direction through the imaging surface 200. In the example of Figs. 2 and 5, the imaging direction would be perpendicular to the imaging surface 200. The sample recess 120 features a lateral enclosing surface 123, which is formed into the body portion 110 to connect the openings 121, 122. The second area A2 defined by the bottom opening 122 is larger than the first area Al of the first opening 121, whereby the lateral enclosing surface 123 features a generally downwardly opening flare. The lateral enclosing surface 123 therefore forms a sample volume V within the body portion 110 between the openings 121, 122. For providing an adequate volume for the three-dimensional sample, the distance between the top and bottom surfaces 111, 112 are set such that preferably a volume of 15 to 100 μΐ is established. In the shown example, the top and bottom openings 121, 122 are circular, whereby the sample recess has a trunco-conical shape. In such a scenario, the diameter of the top opening 121 would be in the range of 1.5 mm to 7 mm, whereas the diameter of the bottom opening 122 would be in the range of 5 to 10 mm. It is to be un- derstood that also other shapes for the openings are feasible. For example, an octagonal shape would achieve the same technical effect. Indeed, even a quadrilateral or a square shape could be conceivable, whereby the resulting sample recess would resemble a pyramid.

The bottom surface 112 of the body portion 110 extends over an area, which is substan- tially larger than the second area A2 of the bottom opening 122 of the sample recess 120 for establishing a support surface large enough to counteract the weight of the sample. If there is a plurality of sample recesses, the bottom surface 112 of the body portion extends over an area, which is larger than the areas covering the bottom openings of the plurality of sample recesses combiner. In either case, the bottom surface 112 preferably provides a surrounding peripheral area of at least 1 mm around the second area A2. Larger surrounding bottom surfaces of 2 to 20 mm, particularly 3 to 10 mm, are also possible for enhancing stabilizing effect of the bottom surface. The ratio of the areas of bottom surface 112 and the second area (A2) needed to achieve the desired support is dependent on material of the sample holder and the size and shape of the sample volume, and weight of the sample. When the material is hard and rigid such as glass, 10% larger bottom surface is enough meaning that the area of the bottom surface 112 is at least 1.1 times the area of the second area A2 alone or combined. When body portion is made of less rigid material, such as silicone elastomer, at least 20% larger area of the bottom surface is preferred. An exemplary body portion is made of PDMS and the bottom surface 112 extends over an area which is at least 20% larger than the second area A2.

In a multiwell configuration as demonstrated by Figs. 3, 7 and 10, the bottom surface 112 extends over an area, which is substantially larger than the area covered by the bottom openings of the sample recesses 120a to 120d or 120L The body portion 110 may be provided with a plurality of such sample recesses, such as anything from one to 20 or even more. According to another embodiment, the body portion 110 also features an additional collar 110a protruding upwards from the top surface 111 for providing an inlet well for e.g. cell culturing medium and/or molecules as shown in Figs. 7 and 10.

Turning now particularly to Figs. 2 and 5, which best shows the angle Θ between the lateral enclosing surface 123 and to the projection of the bottom surface 112 of the body portion 110 along the bottom opening 122. The angle Θ ranges from 30 to 60, more preferably about and including 45 degrees. The angle Θ provides for minimal optimal distortion of the light beam during high resolution microscopy as the lateral enclosing surface 123 is flanked to accommodate the imaging beam, which enters the sample volume 120 through the imaging surface 200. As the lateral enclosing surface 123 is slanted, also the peripheral areas of the second opening 122 may be imaged without any edge disturbances.

A sample holder according to another embodiment the present invention constitutes a cell culture system including a cell culture tray, such as a microtiter plate or cell culture plate, with one or more wells having substantially flat bottom surface. Each such well is con- figured to contain a liquid volume. The sample holder constituting the cell culture system also includes one or more body portions as described above and configured to fit into one or more of the wells of the cell culture tray.

Exemplary sample holders 100 which constitute cell culture systems are shown in Fig. 8 to 10. As shown in these Figs., the body portion 110 is housed in a frame portion 130 of the cell culture tray. The frame portion 130 of the cell culture tray, which includes a transparent bottom wall 132, supports the body portion 110. The body portion 110 has been arranged such that the bottom surface 112 of the body portion 110 mates with an upper surface of the bottom wall 132, and thus the upper surface of the bottom wall constitutes the imaging surface arranged to close the bottom opening 122 of the body por- tion. The cell culture tray further includes an upright wall 131 extending from the bottom wall 132 and surrounding the body portion 110. The body portion 110 has similar constructional features as explained above with reference to Figs. 1 to 3. As shown in Fig. 7, the body portion 110 may house a plurality of sample recesses 120a to 120i, such as nine shown in the Fig. 7, or anything from one to 20 or even more. It is to be understood that the inventive concept also includes a mold, which is configured to produce a sample holder or cell culture system as herein described.

The specific examples provided in the description given above should not be construed as limiting the scope and/or the applicability of the appended claims.

TABLE 1: LIST OF REFERENCE NUMBERS.

Number Part

100 sample holder

110 body portion

110a collar

110b base

111 top surface

112 bottom surface

120 sample recess

121 top opening

122 bottom opening

123 lateral enclosing surface

130 frame portion

131 upright wall

132 bottom wall

200 imaging surface

Al first area

A2 second area

V volume

Θ angle

Claims

1. A sample holder (100) for holding at least one three-dimensional biometric sample, the sample holder (100) comprising:

- a body portion (110) having a top surface (111) and a bottom surface (112), and

- a sample recess (120) formed into the body portion (110) for receiving the sample, the sample recess having (120):

• a top opening (121) formed into the top surface (111) of the body portion (110) and extending over a first area (Al) along the top surface (H I),

• a bottom opening (122) formed into the bottom surface (112) of the body portion (110) and extending over a second area (A2) along the bottom surface (112), and

• a lateral enclosing surface (123) connecting the openings (121, 122) and defining a sample volume (V) between said openings (121, 122) for the three-dimensional sample,

wherein the second area (A2) is larger than the first area (Al), characterized in that the sample holder (100) further comprises an imaging surface (200) arranged to close the bottom opening.

2. The sample holder (100) according to claim 1, wherein the bottom surface (112) of the body portion (110) extends over an area, which is at least 10%, preferably at least 20%, larger than the second area (A2).

3. The sample holder (100) according to claim 1 or 2, wherein the bottom surface (112) extends around the bottom opening (122) so as to provide a surrounding peripheral area of at least 1 mm, particularly 2 to 20 mm, more particularly 3 to 10 mm, around the second area A2.

4. The sample holder (100) according to any of the preceding claims, wherein the second area (A2) is at least 10 % larger than the first area (Al).

5. The sample holder (100) according to any of the preceding claims, wherein the openings (121, 122) are coaxial.

6. The sample holder (100) according to any of the preceding claims, wherein the lateral enclosing surface (123) of the sample recess (120) defines an angle (Θ) of from 30 to 60 degrees respect to the projection of the bottom surface (112) of the body portion (110) along the bottom opening (122).

7. The sample holder (100) according to claim 6, wherein the angle (Θ) is about 45 degrees.

8. The sample holder (100) according to any of the preceding claims, wherein the sample holder (100) comprises a plurality of sample recesses (120a ... 120d), wherein the bottom surface (112) of the body portion (110) extends over an area, which is larger than the combined area covered by the second areas (A2) of the plurality of sample recesses (120a ... 120d), preferably at least 10%, more preferably at least 20% larger.

9. The sample holder (100) according claim 1, wherein the openings (121, 122) are aligned to be parallel with the intended imaging direction through the imaging surface (200).

10. The sample holder (100) according to any of the preceding claims, wherein:

- the body portion (110) is made of a silicone elastomer for attachment to the imaging surface without adhesives, and wherein

- the bottom surface (112) of the body portion (110) extends over an area, which is at least 10% larger than the second area (A2).

11. The sample holder (100) according to any of the preceding claims, wherein body portion (110) comprises a collar (110a), which protrudes upwards from the top surface (111) of the body portion (110) for providing a medium chamber.

12. The sample holder according to any of claims 1 to 10, wherein the sample holder comprises a cell culture tray (130) having at least one well having a substantially flat bottom surface, each well is capable of containing a liquid volume, and the body portion is arranged to fit into the well of the cell culture tray (130).

13. The sample holder according to claim 12, wherein the cell culture tray is a microliter plate or a cell culture plate.

14. The sample holder according to claim 12 or 13, wherein the bottom of the at least one well is optically transparent.

15. The sample holder according to claim 12, 13 or 14, wherein cell culture tray comprises:

- a transparent bottom wall (132) on top of which the body portion (110) has been arranged such that the bottom surface (112) of the body portion (110) mates with an upper surface of the bottom wall (132) and the upper surface of the bottom wall constitutes the imaging surface arranged to close the bottom opening of the body portion, and

- an upright wall (131) extending from the bottom wall (132) and surrounding the body portion (110).

16. A mold having a form suitable for manufacturing, by mold casting, the sample holder according to any of claims 1 to 11.