Positioning device for cell cluster planking
The positioning device used for cell cluster plating solves the problem of uneven cell cluster distribution in iPSC cell culture, achieving uniform deposition and independent growth of cell clusters, improving culture efficiency and consistency, and meeting the requirements of standardized research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- ZHEJIANG TIANYUAN BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-06-05
- Publication Date
- 2026-05-19
AI Technical Summary
In existing iPSC cell culture methods, the cell clusters are unevenly distributed, resulting in local over-density or under-density, which affects cell growth and culture efficiency, increases costs, and may lead to unstable cell states.
A positioning device for cell cluster plating is employed, comprising a base and a support. The base has multiple evenly arranged positioning holes to guide cell clusters to deposit in a designated area at the bottom of the culture container. By precisely controlling the distance and size of the positioning holes, the uniform distribution and independent growth of cell clusters are ensured.
It achieves selective localization and uniform distribution of cell clusters, reduces cell fusion and differentiation, improves experimental reproducibility and consistency, reduces the need for manual operation, and supports efficient amplification and subsequent applications.
Smart Images
Figure CN224258650U_ABST
Abstract
Description
Technical Field
[0001] This utility model relates to the field of biological cell culture technology, and more specifically, to a positioning device for cell cluster plating. Background Technology
[0002] PSC cells are a type of pluripotent stem cell, generally divided into two categories: embryonic stem cells (ESCs) derived from blastocyst embryos and induced pluripotent stem cells (iPSCs).
[0003] With the rapid development of stem cell research, induced pluripotent stem cells (iPSCs) have attracted much attention due to their wide application in regenerative medicine, disease modeling, and drug screening. The culture of iPSCs is a crucial step in realizing their clinical application; however, existing culture methods still face many challenges.
[0004] Conventional iPSC culture methods typically involve random seeding of cells onto a culture dish surface coated with extracellular matrix proteins (such as Matrigel), allowing cells to attach and form colonies. This random seeding method results in uncontrollable cell cluster distribution, making it difficult to achieve uniform cell arrangement and often leading to localized over-density or under-density on the culture surface. Over-density areas inhibit cell proliferation by competing for limited growth resources (such as growth factors and metabolic substrates), while under-density areas waste culture space. This inefficient use of space not only increases culture costs but may also lead to cell instability.
[0005] In existing technologies, the main approach is to optimize colony distribution by seeding methods based on cell density (such as adjusting the initial number of seeded cells). However, this method cannot precisely control the spacing between cell clusters and cannot fundamentally solve the above problems.
[0006] Therefore, it is urgent to solve the problem of uneven cell cluster distribution during pluripotent stem cell culture. Utility Model Content
[0007] The technical problem solved by this invention is the uneven distribution of cell clusters during the culture of pluripotent stem cells.
[0008] To address the aforementioned problems, this invention provides a positioning device for cell cluster plating. The positioning device includes a base and a support that cooperate with each other. The base is disposed within a culture container and supported by the support to form an accommodating gap with the bottom of the culture container. The base has a plurality of evenly arranged positioning holes for guiding the cell cluster located at the upper end of the base to the bottom.
[0009] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: By setting up a positioning device for cell cluster plating, the cell clusters are inoculated in a designated area of the culture container during cell culture, thereby achieving selective positioning of cell clusters and optimizing the growth space. Specifically, the positioning device is detachably placed inside the culture container. By setting multiple evenly distributed positioning holes at the bottom, during the cell cluster inoculation step, cell clusters are added to the positioning holes in sequence, so that the cell clusters fall to the bottom of the culture container and the area corresponding to the positioning holes under the guidance of the positioning holes.
[0010] In one embodiment of this utility model, the center distance between two adjacent positioning holes in the plurality of positioning holes is 2500μm~3500μm.
[0011] Compared to existing technologies, the technical advantages of this solution are as follows: The center-to-center distance between two adjacent positioning wells in multiple positioning wells is determined by the cell growth rate and expansion diameter, ensuring that cells do not contact each other during proliferation and maintain independent growth. This is illustrated by the practical situation in traditional cell culture methods, where the random distribution of cell clusters can easily lead to fusion of adjacent colonies during growth, forming excessively large cell clusters. This fusion increases the contact area between cells, thereby activating differentiation signaling pathways (such as the TGF-β / BMP pathway) and reducing the stemness maintenance capacity of iPSCs. Furthermore, insufficient nutrient and oxygen supply in the central region of fused colonies may further induce self-differentiation, affecting the uniformity of cell quality. By precisely controlling the distance between adjacent positioning wells, a minimum colony spacing is set from the source, preventing boundary contact and fusion of cells during expansion, thus maintaining the independence of each colony.
[0012] In one embodiment of this utility model, multiple positioning holes have the same shape and are equal in size.
[0013] Compared with existing technologies, the technical effects achieved by this solution are as follows: multiple positioning holes are of the same shape and size, which allows cell clusters to be deposited uniformly through multiple evenly distributed positioning holes, improving the reproducibility and consistency of the experiment and meeting the stringent requirements of standardized research procedures and multi-center experiments.
[0014] In one embodiment of this invention, the diameter of the positioning hole is 50µm to 500µm.
[0015] Compared with existing technologies, the technical effects achieved by this solution are as follows: By limiting the diameter of the positioning well, only cell clusters of a certain size are allowed to pass through, ensuring the uniformity of cell cluster growth. Considering the actual situation in traditional cell culture processes, in traditional cell culture methods, randomly distributed cell clusters easily form aggregates of varying sizes after enzymatic digestion during passage. Larger cell clusters may have reduced survival rates due to internal cell apoptosis or differentiation. This heterogeneity further worsens the uniformity of subsequent seeding, creating a vicious cycle and ultimately leading to difficulties in experimental reproducibility and large-scale production. This invention, by setting a positioning well of a specific diameter, filters out larger cell clusters. In some embodiments, cell clusters with a diameter less than 230µm can be screened before seeding iPSCs, further ensuring the uniformity of seeding.
[0016] In one embodiment of this utility model, the positioning hole includes a first liquid inlet hole away from the bottom and a second liquid outlet hole near the bottom and disposed opposite to the first liquid inlet hole; wherein the diameter of the second liquid outlet hole is less than or equal to the diameter of the first liquid inlet hole.
[0017] Compared with existing technologies, the technical effects achieved by this technical solution are as follows: the diameter of the second liquid outlet is smaller than or equal to the diameter of the first liquid inlet, which can be used to guide cell cluster aggregation and control cell cluster deposition.
[0018] In one embodiment of this utility model, the accommodating spacing is 0.5mm to 1.5mm.
[0019] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: by limiting the size of the containment spacing, the distance between the base and the bottom of the culture container is maintained; in some embodiments, the culture container is pre-coated with extracellular matrix and is ready for use after sufficient gelation; by maintaining the distance between the base and the bottom of the culture container in the placement direction, the contact between the base and the extracellular matrix at the bottom can be avoided, thereby destroying the adhesion of the extracellular matrix.
[0020] In one embodiment of this utility model, the base is made of a transparent material.
[0021] Compared with existing technologies, the technical effects achieved by adopting this technical solution are as follows: the positioning device is made of transparent material, which facilitates microscopic observation and cell culture.
[0022] In one embodiment of the present invention, the positioning device further includes a clamping assembly, which is detachably mounted on the positioning device.
[0023] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: the clamping component can be used to remove the positioning device from the culture container; when using the positioning device of this utility model for inoculation, the clamping component is first removed from the positioning device, and after inoculation is completed, the clamping component is then installed on the positioning device, and the positioning device is removed from the culture container by using the clamping component, which helps to maintain the stability of the cell cluster at the bottom of the culture container after inoculation.
[0024] In one embodiment of this utility model, the positioning device includes a frame fixedly connected to the base, the frame having two symmetrically arranged mounting holes, the openings of which are perpendicular to the placement direction; the mounting holes are used to cooperate with the clamping assembly.
[0025] Compared with the existing technology, the technical effect achieved by adopting this technical solution is as follows: when the height of the positioning device in the placement direction is less than or equal to the height of the side wall of the culture container, the positioning device is not easy to remove. By setting two symmetrically arranged mounting holes on the frame of the positioning device, the mounting holes are used to cooperate with the clamping assembly, making it easy to remove the positioning device from the culture container.
[0026] In one embodiment of this utility model, the clamping assembly is an elastic element; the clamping assembly includes a clamping part and mounting parts symmetrically distributed at both ends of the clamping part; the mounting parts are used to mate with mounting holes.
[0027] Compared with the prior art, the technical effects achieved by this technical solution are as follows: the clamping component is an elastic element; the clamping component includes a clamping part and mounting parts symmetrically distributed at both ends of the clamping part; wherein, when the positioning device is removed from the culture container, the clamping part is squeezed to shorten the distance between the two mounting parts; when the distance is less than the distance between the two mounting holes, the mounting parts are aligned with the mounting holes and the squeezing action is released, and the two mounting parts extend into the two mounting holes respectively; the clamping part accepts the removal action away from the placement direction, driving the mounting parts to remove the positioning device from the culture container.
[0028] By adopting the technical solution of this utility model, the following technical effects can be achieved:
[0029] (1) Reduce random differentiation by minimizing physical contact between settlements:
[0030] Random contact or colony fusion between iPSCs often leads to abnormal local microenvironments, activating certain signaling pathways and causing some cells to differentiate prematurely. The positioning device of this invention, by limiting the deposition point of each cell cluster and increasing the distance between colonies, can physically isolate different colonies, reduce information interference and physical stimulation between them, thereby reducing the spontaneous differentiation rate and further enhancing the stability of the culture system;
[0031] (2) Improve repeatability and avoid random adhesion:
[0032] Conventional iPSC seeding methods exhibit significant deposition randomness, especially when dealing with small cell clusters, where the colony placement is uncontrollable, leading to large inter-experimental variability and poor reproducibility. This invention, through precise positioning holes and a defined spacing between the holes and the culture container, ensures uniform deposition of cell clusters at predetermined intervals, significantly improving experimental reproducibility and consistency, and meeting the stringent requirements of standardized research procedures and multi-center experiments.
[0033] (3) Reduce manual operations (such as sorting from settlements):
[0034] In iPSC culture, improper handling during cell expansion often leads to random cell differentiation, requiring manual selection, separation, or removal of abnormal colonies. This process is time-consuming, labor-intensive, and susceptible to subjective factors, reducing experimental efficiency and consistency. This invention, through a three-pronged design of colony size screening, targeted deposition, and attachment guidance, significantly reduces the need for random cell clonal differentiation and manual intervention, improves operational efficiency, reduces human error, and lays the foundation for automated and large-scale culture.
[0035] (4) Achieve uniform colony distribution, supporting efficient amplification and subsequent applications:
[0036] Uniform distribution of iPSC colonies is a key prerequisite for achieving high-quality amplification, precise induction of differentiation, and organoid construction. The positioning device of this invention can rapidly achieve regular arrangement of iPSC colonies in a six-well plate, maximizing the utilization of the Matrigel culture area, ensuring uniform cell attachment and synchronous growth, improving amplification efficiency, ensuring stable cell yield and consistent passage time, and providing highly consistent starting material for downstream applications. Attached Figure Description
[0037] To more clearly illustrate the technical solutions of the embodiments of this utility model, the drawings to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0038] Figure 1 A schematic diagram of the upper position structure of the positioning device provided in this embodiment of the utility model;
[0039] Figure 2 Side view of the positioning device provided in an embodiment of this utility model;
[0040] Figure 3 A schematic diagram of the lower end position structure of the positioning device provided in this embodiment of the utility model;
[0041] Figure 4 for Figure 3 Enlarged view of a portion of point A in the middle.
[0042] Explanation of reference numerals in the attached figures:
[0043] 100. Positioning device; 110. Base; 111. Positioning hole; 120. Support. Detailed Implementation
[0044] To make the above-mentioned objectives, features and advantages of this utility model more apparent and understandable, the specific embodiments of this utility model will be described in detail below with reference to the accompanying drawings.
[0045] See Figure 1 and Figure 2 This utility model provides a positioning device for cell cluster plating. The positioning device 100 includes a base 110 and a support 120 that cooperate with each other. The base 110 is disposed inside a culture container and supported by the support 120 to form an accommodating gap with the bottom of the culture container. The base 110 is provided with a plurality of evenly arranged positioning holes 111 for guiding the cell clusters located at the upper end of the base 110 to the bottom.
[0046] Furthermore, the culture container can be a six-well plate or cell culture dishes of various sizes. By setting a positioning device 100 for cell cluster placement, the cell clusters are seeded in a designated area of the culture container during cell culture, thereby achieving selective positioning of cell clusters and optimizing the growth space. Specifically, the positioning device 100 is detachably placed inside the culture container. By setting multiple evenly arranged positioning holes 111 at the bottom 110, during the cell cluster seeding step, cell clusters are added to the positioning holes 111 in sequence, so that the cell clusters fall to the bottom of the culture container and the area corresponding to the positioning holes 111 under the guidance of the positioning holes 111.
[0047] In some embodiments, using the positioning device 100 of this invention to plate iPSC cell clusters includes the following steps:
[0048] S100. Cell preparation: Gently digest iPSCs and treat iPSCs with cell dissociation buffer (enzyme-free buffer) to digest iPSCs into single cells and small cell clusters, dissociating them into cell clusters of approximately 70 μm in size;
[0049] S200, Size Screening: The cell suspension is passed through a 70μm cell filter to filter and sort cell clusters of the target size (e.g., 70µm).
[0050] S300. Prepare Matrigel-coated six-well plates: Pre-coat with Matrigel or other extracellular matrix, and allow to gel fully before use;
[0051] S400, Placement of positioning device 100: Place the positioning device 100 into the hole of the six-hole plate. The support part 120 will maintain a 1mm accommodating gap with the Matrigel surface to avoid damaging the Matrigel adhesion.
[0052] S500, Controlled Inoculation: Use a pipette to drop the cell cluster onto the positioning well 111 (one drop per well), and gently shake to ensure the cell cluster is evenly distributed into the positioning well 111.
[0053] S600, Culture Maintenance: After the cell clusters settle in the designated area of the six-well plate and adhere to the Matrigel for 5 minutes, slowly and carefully add culture medium, and then place the plate in a 37°C cell culture incubator for amplification, avoiding fusion.
[0054] See Figure 3 and Figure 4 In a specific embodiment of this utility model, the center distance between two adjacent positioning holes 111 is 2500μm~3500μm.
[0055] Furthermore, the center-to-center distance between two adjacent positioning wells 111 is determined by the cell growth rate and expansion diameter, ensuring that cells do not contact each other during proliferation and maintain independent growth. In some embodiments, the distance between two adjacent positioning wells 111 is set to 3000 μm based on the iPSC growth rate and expansion diameter. Considering the actual situation in traditional cell culture, due to the random distribution of cell clusters, adjacent colonies are prone to fusion during growth, forming excessively large cell clusters. This fusion increases the contact area between cells, thereby activating differentiation signaling pathways and reducing the stemness maintenance capacity of iPSCs. In addition, insufficient nutrient and oxygen supply in the central region of fused colonies may further induce self-differentiation, affecting the uniformity of cell quality. By precisely controlling the distance between adjacent positioning wells 111, the minimum colony spacing is set from the source, preventing boundary contact and fusion of cells during expansion, thereby maintaining the independence of each colony.
[0056] In one specific embodiment of this utility model, the multiple positioning holes 111 have the same shape and are equal in size.
[0057] Furthermore, the positioning holes 111 can be circular, square, or other geometric shapes. In some embodiments, the positioning holes 111 are circular. Multiple positioning holes 111 have the same shape and are of equal size, so that the cell clusters are uniformly deposited according to multiple uniformly arranged positioning holes 111, which improves the reproducibility and consistency of the experiment and meets the strict requirements of standardized research procedures and multi-center experiments.
[0058] In one specific embodiment of this utility model, the diameter of the positioning hole 111 is 50µm~500µm.
[0059] Furthermore, by limiting the diameter of the positioning well 111, only cell clusters of a certain size are allowed to pass through, ensuring the uniformity of cell cluster growth. This is illustrated by the practical situation in traditional cell culture processes. Using traditional cell culture methods, randomly distributed cell clusters easily form aggregates of varying sizes after enzymatic digestion during passage. Larger cell clusters may have reduced survival rates due to internal cell apoptosis or differentiation. This heterogeneity further worsens the uniformity of subsequent seeding, creating a vicious cycle that ultimately leads to difficulties in experimental reproducibility and large-scale production. This invention filters out larger cell clusters by setting positioning holes 111 of a certain diameter. In some embodiments, before inoculating iPSCs, cell clusters with a diameter of less than 230µm can be screened to further ensure the uniformity of inoculation. Specifically, before using this positioning device 100, cell preparation is performed by gently digesting iPSCs and using cell dissociation buffer, specifically an enzyme-free buffer, to digest iPSCs into single cells and small cell clusters, dissociating them into small cell clusters. Then, size screening is performed by passing the dissociated cell suspension through a cell filter to filter and sort cell clusters of the target size, specifically 70µm-µm.
[0060] In a specific embodiment of this utility model, the positioning hole 111 includes a first liquid inlet hole away from the bottom and a second liquid outlet hole near the bottom and opposite to the first liquid inlet hole; wherein the diameter of the second liquid outlet hole is less than or equal to the diameter of the first liquid inlet hole.
[0061] Furthermore, the diameter of the second outlet hole is smaller than or equal to the diameter of the first inlet hole, which is used to guide cell cluster aggregation and control cell cluster deposition.
[0062] In one specific embodiment of this utility model, the accommodating spacing is 0.5mm to 1.5mm.
[0063] Furthermore, in some embodiments, the spacing is 1 mm. Referring to actual iPSC culture conditions, the culture container is pre-coated with extracellular matrix and allowed to fully gel before use. By maintaining a 1 mm spacing between the base 110 and the bottom of the culture container in the placement direction, contact between the base 110 and the extracellular matrix at the bottom can be avoided, thus preventing disruption of the extracellular matrix's adhesion. Furthermore, this positioning device 100 utilizes precision micromachining techniques, including photolithography, soft etching, or mold hot pressing, to ensure that the spatial position of each positioning hole 111 is highly consistent, resulting in a constant colony spacing. This standardized structure effectively avoids "hot zones" or "dead zones" caused by random distribution, thereby improving the synchronicity and overall uniformity of colony growth.
[0064] In one specific embodiment of this utility model, the positioning device 100 is made of transparent material.
[0065] Furthermore, the base 110 is made of a transparent material to facilitate microscopic observation and cell culture. In some embodiments, the transparent material can be a transparent polymer, such as polydimethylsiloxane (PDMS), polystyrene (PS), polymethyl methacrylate (PMMA), and glass.
[0066] In one specific embodiment of the present invention, the positioning device 100 further includes a clamping component, which is detachably mounted on the positioning device 100.
[0067] Furthermore, the positioning device 100 also includes a clamping component, which is detachably mounted on the positioning device 100. The clamping component can be used to remove the positioning device 100 from the culture container. When using the positioning device 100 of this invention for inoculation, the clamping component is first removed from the positioning device 100. After inoculation is completed, the clamping component is then mounted on the positioning device 100. The clamping component is used to remove the positioning device 100 from the culture container, which helps to maintain the stability of the cell cluster arrangement at the bottom of the culture container after inoculation.
[0068] In a specific embodiment of this utility model, the positioning device 100 includes a frame fixedly connected to the base 110. The frame has two symmetrically arranged mounting holes, the openings of which are perpendicular to the placement direction. The mounting holes are used to cooperate with the clamping components.
[0069] Furthermore: When the height of the positioning device 100 in the placement direction is less than or equal to the height of the side wall of the culture container, the positioning device 100 is not easy to remove. By providing two symmetrically arranged mounting holes on the frame of the positioning device 100 for cooperation with the clamping assembly, it is easy to remove the positioning device 100 from the culture container.
[0070] In a specific embodiment of this utility model, the clamping component is an elastic element; the clamping component includes a clamping part and mounting parts symmetrically distributed at both ends of the clamping part; the mounting parts are used to cooperate with the mounting holes.
[0071] Furthermore: the clamping assembly is an elastic element; the clamping assembly includes a clamping part and mounting parts symmetrically distributed at both ends of the clamping part; wherein, when the positioning device 100 is removed from the culture container, the clamping part is squeezed to shorten the distance between the two mounting parts; when the distance is less than the distance between the two mounting holes, the mounting parts are aligned with the mounting holes and the squeezing action is released, and the two mounting parts extend into the two mounting holes respectively; the clamping part receives a removal action away from the placement direction, driving the mounting parts to remove the positioning device 100 from the culture container.
[0072] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.
Claims
1. A positioning device for cell cluster plating, characterized in that, The positioning device (100) includes a base (110) and a support (120) that cooperate with each other; The base (110) is disposed inside the culture container and is supported by the support (120) to form an accommodating gap with the bottom of the culture container; The base (110) is provided with a plurality of uniformly arranged positioning holes (111) for guiding the cell cluster located at the upper end of the base (110) to the bottom.
2. The positioning device according to claim 1, characterized in that, The center distance between two adjacent positioning holes (111) is 2500μm~3500μm.
3. The positioning device according to claim 1, characterized in that, The multiple positioning holes (111) are identical in shape and equal in size.
4. The positioning device according to claim 1, characterized in that, The diameter of the positioning hole (111) is 50µm~500µm.
5. The positioning device according to claim 1, characterized in that, The positioning hole (111) includes a first liquid inlet hole away from the bottom and a second liquid outlet hole near the bottom and opposite to the first liquid inlet hole; Wherein, the diameter of the second liquid outlet is less than or equal to the diameter of the first liquid inlet.
6. The positioning device according to claim 1, characterized in that, The size of the accommodating gap is 0.5mm to 1.5mm.
7. The positioning device according to claim 1, characterized in that, The base (110) is made of transparent material.
8. The positioning device according to claim 1, characterized in that, The positioning device (100) further includes a clamping assembly that is detachably mounted on the positioning device.
9. The positioning device according to claim 8, characterized in that, The positioning device (100) includes a frame fixedly connected to the base (110); the frame is provided with two symmetrically arranged mounting holes, the openings of which are perpendicular to the placement direction; the mounting holes are used to cooperate with the clamping assembly.
10. The positioning device according to claim 9, characterized in that, The clamping assembly is an elastic element; The clamping assembly includes a clamping part and mounting parts symmetrically distributed at both ends of the clamping part; the mounting parts are used to mate with the mounting holes.