CN106591119A

CN106591119A - Cell culture device

Info

Publication number: CN106591119A
Application number: CN201510665395.1A
Authority: CN
Inventors: O·艾格勒; S·伍德赛德
Original assignee: StemCell Technologies Inc
Current assignee: StemCell Technologies Inc
Priority date: 2015-10-15
Filing date: 2015-10-15
Publication date: 2017-04-26
Anticipated expiration: 2035-10-15
Also published as: CN106591119B

Abstract

A cell culture device comprises holes. The holes are provided with many micro holes and the first public fluid volume is above the micro holes. Each micro hole includes a group of sub-micro holes and the second public fluid volume is above the group of sub-micro hole in each micro hole.

Description

Cell culture system

Technical field

It relates to cell culture system.Specifically, it relates to can be used for the thin of colony forming assay Born of the same parents' culture apparatuses, such as porous plate.

Background technology

Following content is not to recognize that following public any content is prior art or those skilled in the art A part for common knowledge.

The colony forming cell (CFC) of non-adherent cell is determined and typically makes cell in the fluid stream for preventing convection current Carry out in mobile semi-solid or gel-like media, and therefore limit position of the daughter cell from parental cell Mobile distance.This cause when daughter cell continue divide when from individual cells multicellular colony formation. Colony forming assay can provide the quantitative information with regard to the number of each great-hearted CFU-GM in sample, and And allow to be used for point discrete sampling of each colony sub-clone or further analysis.In stem cell or CFU-GM In the case of, CFC is determined and can also be allowed that colony is classified as into different pedigrees based on morphology.Therefore, CFC is determined can be allowed to carry out the CFU-GM in sample the identification of quantitative and pedigree.

Nanopore device also has been used to CFC measure.Such device be intended to by each cell sequestration it is determined that Sentence the operation and research allowed to them in position.

The content of the invention

There is provided the following content of the invention to introduce ensuing more detail discussion to reader.Present invention is not It is intended to limit or limit claim.

According on one side, cell culture system includes hole.Have multiple micropores in hole, and in hole Micropore top is the first common fluid volume.There can be one group of sub-micro hole in each micropore, wherein at each Described group of sub-micro hole top is the second common fluid volume in micropore.

The hole can be limited at least in part by least one hole side wall and bottom hole wall.Each micropore can be with At least in part by limiting from the upwardly extending at least one microporous side wall of the bottom hole wall.Each sub-micro hole can With at least in part by limiting from the upwardly extending at least one sub- microporous side wall of the bottom hole wall.Each sub-micro Hole further can be limited by a part for a microporous side wall in the microporous side wall.

The bottom hole wall can be transparent or translucent.

Every group of sub-micro hole can include the 4 sub-micro holes arranged with 2x2 arrays.In alternative example, Sub-micro hole is with the array arrangement of another kind of construction such as 2x1,3x1,3x2,3x3 or bigger.

Each sub-micro hole can be comprising sub-micro hole top and sub-micro bottom hole portion, and each sub-micro hole can be in horizontal stroke It is gradually reduced to sub-micro bottom hole portion from sub-micro hole top on sectional area.For example, each sub-micro hole can be butt It is taper or frustum.

Each micropore can be comprising micropore top and micropore bottom, and each micropore can be on cross-sectional area Taper into micropore bottom from micropore top.For example, each micropore can be frustoconical or frustum Body.

Sub-micro hole, micropore and hole may be integrally formed.

Cell culture system can include the magnetic or magnetizable element being positioned at below sub-micro hole.Magnetisable unit Part can be wiregrating.Hole can be limited at least in part by bottom hole wall, and wiregrating can be embedded in bottom hole wall It is interior.

Each micropore can have micropore top and relative micropore bottom, and the top of each micropore can be with With at least 100 microns of pore widths.Each micropore micropore depth between the top and the bottom can be At least 75 microns.

Each micropore can have micropore top and micropore bottom.Each micropore may be embodied at micropore top Full-size and micropore top and micropore bottom between micropore depth.The full-size is micro- with described The ratio of hole depth can be 1.1: 1 to 1.9: 1.

According on the other hand, cell culture system includes hole.There are multiple micropores in hole.Each is comprising micro- Hole top and micropore bottom.Each micropore be included in micropore top place full-size, and micropore top with Micropore depth between micropore bottom.The ratio of the full-size and the micropore depth be for about 1.1: 1 to 1.9∶1。

Full-size can be at least 140 microns.Micropore depth can be at least 75 microns.

Each micropore can be tapered into from the top of micropore on cross-sectional area to micropore bottom.For example, each Micropore can be frustoconical or frustum.

The hole can be limited at least in part by least one hole side wall and bottom hole wall.The bottom hole wall can Being transparent or translucent.Each micropore can at least in part by upwardly extending at least from bottom hole wall One microporous side wall is limited.

Cell culture system can further comprising the magnetic or magnetisable grid being positioned at below micropore.Kong Ke To be limited by bottom hole wall at least in part, and grid can be embedded in bottom hole wall.

Micropore and hole may be integrally formed.

Cell culture system can be further contained in hole the first common fluid volume above micropore.

Cell culture system can further comprising one group of sub-micro hole in each micropore.Every group of sub-micro hole can be with Comprising the 4 sub-micro holes arranged with 2x2 arrays.

The hole can be limited at least in part by least one hole side wall and bottom hole wall.Each micropore can be with At least in part by limiting from the upwardly extending at least one microporous side wall of bottom hole wall.Each sub-micro hole can be down to Partially by limiting from the upwardly extending at least one sub- microporous side wall of bottom hole wall.Each sub-micro hole can be entered One step is limited by a part for a microporous side wall in microporous side wall.

Sub-micro hole can be integrally formed with micropore and hole.

It is second public that cell culture system can be further contained in each micropore above described group of sub-micro hole Common fluid volume.

In one embodiment, there is provided cell culture system, it is included：

The hole limited by least one hole side wall and bottom hole wall；

Multiple micropores in the hole, each micropore from the bottom hole wall by upwardly extending and by pore fluid It is described micro- at least one microporous side wall that volume separates with the pore fluid volume of adjacent cells and the hole The first common fluid volume defining above hole, each micropore includes micropore top and micropore bottom, and its In microporous side wall between adjacent micropore converge to form summit；With

The second public stream in one group of sub-micro hole and each micropore in each micropore above described group of sub-micro hole Body volume, one group of sub-micro hole is used to accommodate one or more cells in each sub-micro hole, each sub-micro Hole includes sub-micro hole top, sub-micro bottom hole portion and upwardly extending by sub-micro hole fluid volume from the bottom hole wall With at least one sub- microporous side wall that the sub-micro hole fluid volume in adjacent sub-micro hole separates.

In one embodiment, each sub-micro hole is further by a microporous side wall in the microporous side wall A part restriction.In one embodiment, the bottom hole wall is transparent or translucent.

In one embodiment, every group of sub-micro hole includes the 4 sub-micro holes arranged with 2x2 arrays.One In individual embodiment, every group of sub-micro hole includes the Asia arranged with the array of 2x1,3x1,3x2,3x3 or bigger Micropore.

In one embodiment, each sub-micro hole on cross-sectional area from the top of the sub-micro hole to the Asia Micropore bottom is gradually reduced, and optionally, wherein each sub-micro hole is frustoconical or frustum. In one embodiment, each micropore on cross-sectional area from the top of the micropore to the micropore bottom gradually Diminish, optionally wherein each micropore is frustoconical or frustum.

In one embodiment, one or more in sub-micro hole, micropore and hole are integrally formed.

In one embodiment, cell culture system includes the magnetic or magnetisable being positioned at below sub-micro hole Element.Optionally, magnetizable element is the wiregrating being embedded in bottom hole wall.

In one embodiment, micropore top has at least 100 microns of width.In an embodiment In, the width at the top of micropore is at least 250 microns, at least 400 microns, at least 500 microns or about 50 Micron is to 1000 microns.In one embodiment, each micropore depth between the top and the bottom be to It is few 25 microns.In one embodiment, each micropore depth between the top and the bottom is at least 75 Micron, at least 100 microns, at least 200 microns, at least 300 microns or about 25 microns to 900 microns.

Optionally, and/or additionally, in one embodiment, each micropore is included at micropore top Full-size and micropore top and micropore bottom between micropore depth, and the full-size with it is described The ratio of micropore depth is between 1.1: 1 to 1.9: 1.In one embodiment, full-size is at least 140 Micron.In one embodiment, micropore depth is at least 75 microns.

In one embodiment, microporous side wall is less than 30 degree relative to the angle of vertical direction.At one In embodiment, microporous side wall is less than 20 degree relative to the angle of vertical direction.

Any feature as herein described or combinations of features are included, if as based on context, this explanation The knowledge of book and those of ordinary skill in the art will be apparent, the spy that any such combination includes Levy not inconsistent mutually.

Description of the drawings

In a specific embodiment reference is carried out to accompanying drawing, in the accompanying drawings：

Figure 1A is the image of the colony being typically observed in hematopoietic colonies are determined, it is shown that many has big The colony of degree of overlapping is individual；

Figure 1B is the image of the colony being typically observed in hematopoietic colonies are determined, it is shown that from single With multiple centers colony of ancestors；

Fig. 2 is the perspective view of the exemplary cells culture apparatuses of the disclosure；

Fig. 3 is the top view of the cell culture system of Fig. 2；

Fig. 4 is the zoomed-in view in the region in Fig. 3 shown in box 4；

Fig. 5 is the cross section taken along Fig. 4 center line 5-5；

Fig. 6 is the perspective cut-away schematic view in the region in Fig. 3 shown in box 4；

Fig. 7 is the zoomed-in view in the region in Fig. 5 shown in circle 7, it is shown that in being embedded in bottom hole portion Magnetisable grid；

Fig. 8 shows the image by the multiple microporous structures acquired in bright-field microscope；

Fig. 9 A to 9C are the immobilized images for showing fluorescent bead in micropore；

Figure 10 A to 10E are the images for showing the example of Colony forming in different microporous structures；

Figure 11 A are to the image acquired in cell culture system, the cell culture by bright-field microscope Device includes the sub-micro hole in micropore and micropore；

Figure 11 B are the images obtained to cell culture system using colored CCD scanner, the cell culture Device includes the sub-micro hole in micropore and micropore；

Figure 12 A to 12C (are carried using ccd line sensor12- line color sensors Epson V500 scanneies) obtain the cell culture system comprising micropore in dyeing colony image；

Figure 13 is to show counting and standard to the hepatocyte progenitors in the cell culture system comprising micropore The figure of the dependency between CFC measure；

Figure 14 is to show the base when multiple colony forming cells of each micropore inoculation are corrected using Poisson distribution The figure of the range of linearity of the increase determined in the CFC of micropore；

Figure 15 A are to the image acquired in the micropore containing magnetic microcarrier pearl by bright-field microscope；

Figure 15 B are to the image acquired in the micropore of Figure 14 A after incubation 7 days by bright-field microscope；

Figure 16 is to show that the sedimentation that magnetic force is aided in does not affect the Colony forming being based on during the CFC of micropore is determined Figure.

Specific embodiment

Multiple devices or method is described below to provide the reality of the embodiment of each claimed invention Example.Embodiments described below does not limit any claimed invention, and any claimed sends out It is bright to cover the following method or apparatus without description.Claimed invention is not limited to following described Any one device or method the common spy of all features or multiple devices described below or all devices The device levied or method.It is possible that devices explained below or method are not by present patent application The embodiment of any patent rights authorized.Appointing disclosed in the device or method described in below Any invention what invention authorizes patent rights with the mandate for not passing through present patent application can be another protectiveness File for example continues the theme of patent application, and applicant, inventor or everyone be not intended to abandon any The invention, any invention of exclusion contribute any institute by the disclosure in its presents to the public State invention.

Semi-solid medium may be determined to quantitative or sub-clone the CFC for each cell has some limits System.Because cell is without firmly fixing in media as well, operation processes vessel and may interfere with colony.For example, Such as continually move culture dish or the disturbance to culture adding liquid reagent potentially interferes with colony.This can The type and number of times of the operation that can be carried out to these cultures can be limited.

Further, since without physical boundary in culture, some colonies may be overlapped, making it difficult to it is determined that Whether neighbouring cell mass is derived from the colony of single CFU-GM, or whether represents the list with multiple centers Individual colony, the plurality of center is by from one section of short-range daughter cell generation of primitive progenitors migration. This can cause the error count to total colony number.

Additionally, the morphological feature Jing of the colony of different mature cell pedigrees is often not unique enough so as to this The classification of a little colonies can become the often subjective process with highly variable.For example to hemopoietic progenitor cell In the case that CFC is determined, it may be difficult to distinguish thin from granulocyte ancestors, mononuclear cell ancestors and macronucleus The colony of born of the same parents ancestors, only can allow reliably to distinguish main pedigree type (red system and medullary system).In order to reliably Colony is sorted out according to the type of its CFU-GM originated, it may be necessary to specific labelling and colouring method. Such method often relies on the probe point for introducing identification specificity cell surface marker or cellular content Son.Colouring method typically relates to the fixation to cell and a series of subsequent washings, dyeing and decoloring process. These methods determine incompatible with semi-solid medium to the colony of non-adherent cell, because fixed, dyeing Addition with wash solution can disturb colony.

Another restriction that standard CFC in semi-solid medium is determined is that multiple colonies occur in another colony Neighbouring or appearance has the multiple colonies for overlapping border, as shown in Figure 1A.Such colony be likely difficult to Multicenter colony from individual cells is mutually distinguished, as shown in fig. 1b.This is not only resulted in colony count High subjective analysis, and because the presence of the foreign cell from neighbouring colony causes to be carried from culture Each colony is taken for further expanding or sub-clone becomes complexity.

Although overcoming some shortcomings above-mentioned using known nanopore device, in known micropore dress In putting, colony Jing is often spread to outside the volume of micropore.This causes to spread in adjacent micropore, and/or Wash the cell in colony in conventional processing procedure off.Additionally, in known nanopore device, more than one It is very common that CFU-GM is inoculated in each micropore, is thus caused in each micropore more than a colony Growth.This may cause to be made a mistake in colonies.

It relates to the cell culture system comprising micropore.The cell culture system can be used for Colony forming Determine, and some or all shortcomings above-mentioned can be overcome.Especially, as described in further detail below Ground, cell culture system as described herein preferably by each colony compartmentation can be born in collection Preferably them are caused without departing from the volume of micropore, and can cause when long comprising the receiving colony Multicellular colony is steadily captured in the culture of many days, to prevent in operation such as addition or during removing solution Or reduce the chance that they spread between micropore or spread.This can allow cell culture system for continue The simple quantitative colony forming assay of about 4-20 days.Additionally, this can be by colony and their nearest neighbours Separate and reduce the appearance of cell overlap, enabling objective counting is carried out to colony and each collection is extracted The pollution fallen without unrelated cell.

In addition, as described herein cell culture system can effectively immobilized cell, thereby eliminate The needs of sample are fixed before dyeing.This can cause sensitive living cells colouring method (for example, to drop into collection Row dyeing is used to reliably sort out colony type) it is possibly realized, the living cells colouring method does not change The metabolism of cell and physical features.In combination with specific cell dyeing method, the colony in micropore can return Class is into hypotype (for example, for hematopoietic colonies：Red system, medullary system, granulocyte, megalokaryocyte, mononuclear cell etc.). Additionally, cell culture system as herein described allows to be imaged cell.

With reference to Fig. 2 and 3, it is shown that exemplary cells culture apparatuses 100.Cell culture system 100 is comprising extremely A few hole 102.As used herein, the cell that term " hole " is typically referred in fluid medium can be placed In any fluid reservoir of the culture for being wherein used for cell.In shown example, cell culture system 100 For the form of porous plate, and comprising 6 holes 102.In alternative example, the cell of multiwell plate format Culture apparatuses can include the hole of alternative number, such as 24 holes or 96 holes.The hole of porous plate is in horizontal stroke Rectangle being typically round on section rather than as shown in Figure 2.However, with generally flat bottom Any container in portion is acceptable.In further alternative example, cell culture system can be located In another kind of suitable form, such as Tissue Culture Dish (as used in examples below chapters and sections).So Example in, cell culture system can include only one hole (that is, the single fluid reservoir of Tissue Culture Dish).

With reference to Fig. 2 and 5, each hole 102 includes hole top 104 and bottom hole portion 106.Additionally, each hole 102 Limited by least one hole side wall 108 and bottom hole wall 110.

Hole can have any suitable shape.In shown example, hole 102 is substantially square, And limited by 4 holes side wall 108 and bottom hole wall 110.Additionally, hole can have it is any suitably sized. For example, hole can have the volume of about 30 μ L- about 10L.For example, the scope in a hole of 384 orifice plates Lower limit is about 30 μ L, and the upper limit of the scope of typical 6 orifice plate is about 100mL.For Qtray, volume is About 1.1L, for the flat board of filling the taking up room of standard incubator shelf (footprint), volume can be as high as 10L.Therefore, in one embodiment, hole has the volume of about 30 μ L- about 10L.In another reality In applying scheme, hole has the volume of about 30 μ L-100mL.In another embodiment again, hole has about The volume of 30 μ L- about 6mL.

Referring now to Fig. 4-6, there is the first of multiple micropores 112 and the top of micropore 112 in each hole 102 Common fluid volume 114 (shows in Figure 5).Specifically, bottom hole portion 106 of the micropore 112 in each hole Place, and separate with hole top 104.It is public that space at 104 at the top of the hole in each hole 102 forms first Common fluid volume 114, it is connected with each micropore 112.

Micropore 112 provides each CFU-GM and can be inoculated with the volume that colony is grown in the inner and in it.That is, Fluid medium containing CFU-GM can be placed in each hole 102.Then can be to cell culture system 100 centrifugations, so as to force CFU-GM to reach the bottom hole portion 106 in each hole 102 and enter in micropore 112, make Obtain each cell to be split in micropore 112 and can grow to form colony.It is alternatively possible in gravity In the presence of make cell settlement into micropore 112.First common fluid volume 114 is allowed in given bore 102 Each micropore 112 share common medium so that the cell in micropore 112 is trained under conditions of being substantially the same Support.

With reference to Fig. 5 and 6, each micropore 112 includes micropore top 116 and micropore bottom 118.Each micropore 112 by the top of micropore between 116 and micropore bottom 118 extend at least one microporous side wall 120 and Microvia bottom 122 at micropore bottom is limited.

With reference to Fig. 5, in shown example, the microvia bottom of each micropore 112 in given bore 102 122 are formed by the bottom hole wall 110 of given bore 102.Additionally, microporous side wall 120 is from the one of bottom hole wall 110 Upwardly extend, and the microporous side wall 120 of the micropore 112 of adjacent bores side wall 108 and hole side wall 108 1 Body ground is formed.

In alternative example, bottom hole wall and hole side wall can separate landform with microvia bottom and microporous side wall Into.For example, micropore can be formed as insert, and the insert is positioned on the diapire in hole and optional Ground is fixed on the diapire (described in following article embodiment chapters and sections) in hole.

Micropore can have any suitable shape.With reference to Fig. 4, in shown example, 116 at the top of micropore It is substantially square to locate each micropore 112, and is limited by 4 microporous side walls 120.Additionally, reference Fig. 5 and 6, each micropore 112 116 gradually subtracts from the top of micropore on cross-sectional area to micropore bottom 118 It is little.More specifically, in shown example, each micropore 112 is substantially frustum.This shape is led to CFU-GM is often promoted to be seeded in micropore 112, rather than between adjacent micropore 112.More specifically, In shown example, because micropore 112 is substantially frustum, the microporous side wall of adjacent cells 112 120 join at summit 124 so that cell generally can not be seeded between micropore 112.

116 reality being gradually reduced to micropore bottom 118 from the top of micropore on cross section of micropore 112 wherein In example, microporous side wall 120 can appoint relative to the angle (referred to herein as " wall angle ") of vertical direction What suitable angle.In some instances, the angle can be less than about 30 degree, such as 10 degree to 20 degree. In other instances, the angle can be as small as 2 degree.As shown in embodiment chapters and sections below, Dang Bijiao During reduction, the volume of micropore increases, and causes to accommodate cell colony increase.

In shown example, microporous side wall 120 extends to micropore with identical angle from micropore top 116 Bottom 118.In alternative example (not shown), microporous side wall can include Part I and Part II, The Part I is extended with first angle from micropore top down, and the Part II is with second angle from institute State Part I to extend downwardly.Second angle can be less than first angle (for example, second angle can be 0 degree). This can allow the microporous side wall of adjacent micropore to join in apex, while it is relative to still allow for microvia bottom Greatly, and micropore volume it is relatively large.

In alternative example (not shown), each micropore place at the top of micropore can be substantial circular, and And can be substantially frustoconical.In another alternative example again, each micropore is at the top of micropore Place can be another suitable shape, such as triangle, rectangle, trapezoidal or hexagon.

In shown example, the micropore 112 in given bore 102 generally has the same shape and dimensions. In alternative example (not shown), the micropore in given bore can have different shape and size.

In shown example, each micropore 112 has substantially in the axis of symmetry in central authorities.In shown example In (not shown), one or more micropores can not have central symmetry axle.

Generally, the colony for being formed in micropore can have about 10-100, the average-size of 000 cell；So And, some colonies can be grown to more than 1,000,000 cells.The cell collection of expected 1,000,000 cells Fall the volume with about 1.0 μ L.Because the micropore in known nanopore device is generally not intended to for cell training Support, therefore for the overwhelming majority, their size is not formed into and accommodates the big colony for occurring once in a while. However, in a known nanopore device, the volume of micropore is for about 0.1 μ L or bigger (Ungrin WO 2008/106,771), it can accommodate these big colonies.As described above, in these nanopore devices, cell Often still can be disseminated to outside the volume of micropore.Astoundingly, have been determined by present customizing micropore Size causes its full-size at top and the ratio of its depth (to be hereinafter also referred to as " full-size depth Degree ratio ") be, less than 1.9: 1, and more particularly, to be 1.9: 1 to 1.1: 1, it is possible to reduce cell dispersion is extremely Outside micropore, and the immobilization to larger colony can be realized.

For example, referring to Fig. 6, each micropore 112 116 at the top of the micropore at be substantially square, and have There are pore widths 101 and microcell length 103 at top 116 and 116 and micropore bottom at the top of micropore Micropore depth 105 between 118.Because micropore 112 is substantially square at top 116, therefore across top The full-size in portion is diagonal 107.Therefore, if micropore depth 105 is of about 1mm, in order to have Full-size depth ratio less than 1.9: 1, the length of line 107 will be less than 1.9mm.For example, pore widths 101 can be 1mm, and microcell length 103 can be 1mm so that the length of line 113 is of about 1.4 mm。

In alternative example, pore widths, microcell length and micropore depth can be another size.Example Such as, pore widths and microcell length can be usually 100 microns or bigger, and more specifically 500 microns Or it is bigger, and typically 75 microns or bigger of micropore depth.Wherein micropore is at top pros Shape and pore widths at top and microcell length be in 500 microns of example, maximum at top Size will be about 707 microns.In such example, in order to have the full-size depth less than 1.9: 1 Than then micropore depth will be greater than about 372 microns.

In alternative example (not shown), wherein micropore has different shapes, across the maximum chi at top Very little can be another size.For example, if micropore at top for circle, full-size will be for top at Diameter.

In each hole the density and total number of micropore can according to the size in the size and shape of micropore and hole and Shape and change.In some instances, the density of micropore can be every square millimeter of 0.5-4.0 in each hole Micropore.In a specific example, each hole can include about 960 micropores.

Referring still to Fig. 4-6, in shown example, there is one group of He of sub-micro hole 126 in each micropore 112 Second common fluid volume 128 of this group of sub-micro hole 126 top in each micropore 112.

As described above, in known nanopore device, it is inoculated in each micropore more than a CFU-GM It is very common, thus causes in each micropore more than the growth of a colony.By in each micropore 112 One group of sub-micro hole 126 of middle offer, is seeded in each micropore 112, CFU-GM even more than a CFU-GM Would generally be divided into adjacent sub-micro hole 126 and be grown into single colony.

Additionally, as described in greater detail below, the size in sub-micro hole 126 can be made for accommodate Average colony (for example, up to 100, the colony of 000 cell), rather than big colony.Therefore, it is sub- Micropore 126 by with enough sizes accommodating the most cells colony of growth；If however, in sub-micro Really big colony is grown in hole 126, then the big colony can grow to the second common fluid volume 128 In and will be comprised in the micropore 112 for accommodating sub-micro hole 126.

In addition, by providing sub-micro hole 126 in each hole 112, the cell from microcolony can be concentrated on At microvia bottom 122.This can strengthen the ability that microcolony is detected by bright-field microscope.For example, this Can be by detecting that colony realizes that faster colony is determined or can detected with relatively low in time point earlier The CFU-GM of multiplication potentiality.

With reference to Fig. 5 and 6, each sub-micro hole 126 includes sub-micro hole top 130 and sub-micro bottom hole portion 132.This Outward, each sub-micro hole 126 is by least extended between 130 and sub-micro bottom hole portion 132 at the top of sub-micro hole Individual sub- microporous side wall 134 and the sub- microvia bottom 136 at sub-micro bottom hole portion 132 are limited.

Sub-micro hole 126 can have any suitable shape.In shown example, each sub-micro hole 126 It is substantially square at 130 at the top of the sub-micro hole, and is limited by 4 sub- microporous side walls 134.

With reference to Fig. 5, in shown example, the sub- microvia bottom in each sub-micro hole 126 in given bore 102 136 are formed by the bottom hole wall 110 of given bore 102.Additionally, in 4 sub-micro holes in each sub-micro hole 126 In side wall 134, two are formed by a part for microporous side wall 120, and two other is from the one of bottom hole wall 110 Upwardly extend.

In alternative example (not shown), micropore diapire, microporous side wall, sub- microvia bottom and sub- microporous side Any one in wall can be formed by single material.For example, the sub-micro hole of given group can be used as insert Formed, the insert is positioned in the microvia bottom of a micropore.

Referring also to Fig. 5 and 6, each sub-micro hole 126 is on cross section from the top of sub-micro hole 130 to sub-micro hole Bottom 132 tapers into.More specifically, in shown example, each sub-micro hole 126 is substantially butt Cone.Similar to micropore 112, this construction promotes CFU-GM to be seeded in sub-micro hole 126, rather than Between adjacent sub-micro hole 126.More specifically, being substantially butt in sub-micro hole 126 in shown example Cone when, the sub- microporous side wall 134 in adjacent sub-micro hole 126 is joined on summit 138 so that cell lead to Often it is not seeded between sub-micro hole 126.

In alternative example (not shown), each sub-micro hole place at the top of sub-micro hole can be substantial circular , and can be substantially frustoconical.In another alternative example again, each sub-micro hole exists Sub-micro hole can be another suitable shape at top, such as triangle, rectangle, trapezoidal or hexagon.

In the example that wherein sub-micro hole tapers into from the top of sub-micro hole on cross section to sub-micro bottom hole portion, Sub- microporous side wall can be any suitable angle relative to the angle of vertical direction.In some instances, institute Stating angle can be less than about 30 degree, such as 10 degree -20 degree.In other embodiments, the angle can be low To 2 degree.

As described above, the size in sub-micro hole 126 can be customized to accommodate average colony.For example, sub-micro Hole can have about 3x 10^-6The volume of the μ L of μ L to about 1.0.For example, referring to Fig. 5, sub-micro hole 126 is on top Width 109 at portion can be about 30 μm to 1mm, and depth 113 between the top and the bottom can be About 30 μm to 1mm.Additionally, sub- microporous side wall 134 can be from vertical direction with the angle of about 1 to 37 degree Degree extends.

Each micropore 112 can include the sub-micro hole 126 of any suitable number and arrangement mode.Shown In example, each micropore 112 includes 4 sub-micro holes 126, and 4 sub-micro holes 126 are with 2x2 arrays row Row.In alternative example, sub-micro hole can with other construction such as 2x1,3x1,3x2,3x3 or Bigger array arrangement.

Referring also to Fig. 5 and 6, bottom hole wall 110 also forms microvia bottom 122 and sub- microvia bottom 136, and it can Being translucent or transparent.This can allow the cell colony in observation of cell culture apparatuses 100, example Such as by microscope or other visual imaging methods.In shown example, sub- microvia bottom 136 is substantially Flat, each sub- microvia bottom 136 is general coplanar.This can aid in examining under a microscope cell Colony.However, in alternative example (not shown), sub- microvia bottom can have another shape, such as Circular.In another alternative example (not shown), sub-micro hole can be not comprising sub- microvia bottom.For example, Sub- microporous side wall can join in apex.

In some example (not shown)s, microvia bottom and/or sub- microvia bottom can be comprising demarcation line as true Determine the mark of the position in micropore or sub-micro hole in cell culture system.

In other example (not shown)s, the inner surface of cell culture system can be applied coated with hydrophobic coating.As general Hydrophobic coating can minimize or reduce the formation of meniscus when liquid is placed in cell culture system, and this can be with Promote being uniformly distributed for the sample being placed in cell culture system.

In other example (not shown)s, the inner surface that can process cell culture system is micro- to promote moistening to cause Hole and sub-micro hole are easily filled with liquid.

As described above, in some instances, cell can be seeded to micropore 112 by centrifugation or by gravity In sub-micro hole 126.In alternative example, it is possible to use cell is seeded to micropore 112 by magnetic force In sub-micro hole 126.For example, in use, cell can be with magnetic particle such asParticle or Other magnetic particle labellings.Particular cell types interested can be used to the cell surface mark on target cell Active substance part (such as glucosan) on the special mixtures of antibodies of will thing and carrier particle and magnetic grain Son is coupled.Using this mixture, antibody complex is formed, the antibody complex is by target cell and magnetic Particle is cross-linked to form magnetic particle, magnetic particle and the target cell complex and unconjugated non-target of suspension The mixture of cell three.Then cell suspension can be deposited in hole 102, and can be subjected to Magnetic field gradient on the direction of bottom hole wall 110.Particle will move up and be gathered in Asia in the side of the gradient Micropore bottom 132.Still remaining in the unwanted cells in suspension can be washed off from hole 102 so that only Leave target cell to form colony in subsequent incubation period.

Referring now to Fig. 7, in some instances, cell culture system can be comprising below sub-micro hole Magnetic or magnetizable element, to increase magnetic field gradient, and improve speed of the particle accumulation in sub-micro bottom hole portion. In shown example, magnetizable element includes magnetisable wiregrating 140, and it is embedded in bottom hole wall 110. Magnetic field gradient can magnetize wiregrating 140, and increase magnetic field gradient.

In alternative example (not shown), wiregrating may be configured to be attracted to the element of magnetic mark often Ad-hoc location in individual hole.

Embodiment

The production of embodiment 1- cell culture system

Prepare in culture dish cell training as above by the way that micropore to be fabricated to insert and insert them into Foster device.Some micropores include sub-micro hole, and some then do not include.Micropore is made for various maximum chis Very little depth ratio, as mentioned above.

The former of several microporous structures is produced by the CNC machinings to solid aluminium dish.These circular dies A diameter of 35mm, thickness is 20mm, and shows the surface of the contrary topological structure with micropore. To be cast in a mold based on the elastomer of polydimethylsiloxane (PDMS), and be subsequently cured elastomer to be formed Flexible disk containing micropore, so as to producing microporous insert.Specifically, by SylGard 184 (DowCorning) 10: 1 (w/w) uniform homogeneous blends of elastomer and firming agent are preparing the elastomer.Pouring Before being cast into aluminum die, the mixture of this silicone component is exposed to vacuum (＜ 10mTorr, 1 hour) to remove Any volatile component.By 1.5g-1.7g elastomers lentamente topple on the mold surface and make its spread with The layer with uniform thickness is formed on mould.Then the pedestal of mould is placed in and is heated on 180 DEG C of hot plate. Due to the minimum thickness and the high conductance of aluminum of mould, to the flash heat transfer of die surface silicone bullet is caused The fast setting of gonosome.After in the period of 5min is heated, mould is removed and by briefly placing from hot plate Ambient temperature is cooled to being cooled on 0 DEG C of aluminium sheet.By an edge for having gently pulled on casting The PDMS gel demouldings of hardening are made to remove the disk containing micropore.

Being inserted into before 35mm culture dishs (Becton-Dickinson, 35-1008) by xeothermic, (135 DEG C are held It is continuous 1 hour) microporous insert is sterilized.By the above-mentioned SylGard elastomers and firming agent of a 125 μ L of drop Mixture is incorporated into the central authorities of the culture dish, then aseptically microporous insert is inserted into into the culture In ware, and array surface is set to face up, so as to microporous insert is bonded in the culture dish.By should Culture dish incubates (2-4 hours) with the bonding of heat cure SylGard at 80-85 DEG C in an oven with array interposer Layer, so as to insert is sealed into hole.

The example of some microwell arrays for producing in this way shows that in fig. 8 it is micro- by the bright visual field The top view image of the micropore of the insert acquired in mirror.The size of micropore is shown in following table, most Large scale depth ratio is shown as the ratio of maximum horizontal size and depth：

Construction	Wall angle (degree)	Pore widths (mm)	Micropore depth (mm)	Full-size depth ratio
					A	37	0.8	0.38	3.0∶1
B	20	0.8	0.5	2.3∶1
					C	15	0.8	0.5	2.3∶1
D	15	1.0	1.0	1.4∶1

Embodiment 2- hole constructs the immobilized effect to particle

Evaluate the ability of the cell culture system compartmentation particle with various sizes of micropore.In this embodiment The cell culture system of middle test does not contain sub-micro hole.Using fluorescence ps particle (Bangs FS06F, 7.3 μ m diameter) complete to evaluate.The suspension of microgranule is placed in the number of individual micropore of several cell culture systems.Institute There is micropore to be all foursquare at the top of micropore.The micropore has following size：

Construction	Wall angle (degree)	Pore widths (mm)	Micropore depth (mm)	Full-size depth ratio
					A	15	1.0	1.0	1.4∶1
B	15	0.8	0.5	2.3∶1
					C	37	0.8	0.38	3.0∶1

Make particle by gravitational settling into hole；Remaining hole is vacant, only containing phosphate buffer (PBS). Then cell culture system is made to receive to represent the Physical Interference method of the operation for being typically used for cell culture application.Tool Body ground, by removing PBS above and replacing carries out cleaning step with the fresh PBS of 2.0mL volumes. Then cell culture system is made to receive quickly lateral (from one side to another side) mobile.Observation is in the week of hole containing particle Whether occur being spread between the Kong Yukong of particle in the micropore for enclosing, and by fluorescence microscope (Leica DMIL Inverted microscope, 4x object lens) it is imaged.The image in the hole around hole containing particle shows in fig .9.

In fig .9, first image in each column is that the center that fluorescent bead is placed on into cell culture system is attached The bright field view of micropore after near micropore.Next image in each column is displayed in cell culture system The fluorescence observed in micropore before any operation.Last image in each column is displayed in cleaning and thing The fluorescence observed in micropore after reason migratory cell culture apparatuses.

Do not see particle transfer occur in the image obtained using fluorescence microscope after the activation.This display Micropore with 1.4: 1 to 3.0: 1 full-size depth ratio is effectively limited during regular growth culture operation The movement of small particles is made.

Embodiment 3- hole constructs the impact to accommodating cell colony

Hematopoietic progenitor cells in liquid medium within are seeded to into the cell containing the micropore with various constructions In culture dish.The cell culture system tested in the present embodiment does not contain sub-micro hole.All micropores are in micropore Top is foursquare.The size of micropore is summarized in the following table：

Construction	Wall angle (degree)	Pore widths (mm)	Micropore depth (mm)	Full-size depth ratio
					A	37	0.8	0.38	3.0∶1
B	30	1	0.75	1.9∶1
					C	20	1	0.75	1.9∶1
D	15	1	0.75	1.9∶1
					E	15	1	1	1.4∶1

With about 7 colony/cm²Colony density inoculating cell culture apparatuses and by slow speed centrifugation by cell In being settled down to micropore.The cell culture system of inoculation subsequently 37 DEG C license culture environment and contain 5%CO₂ Humidifying atmosphere in be incubated.With the time interval monitoring Colony forming of two days and the micropore of cell is recorded to micro- Any evidence that hole spreads.

Figure 10 shows the feature of the Colony forming in 5 kinds of microporous structures.

With regard to constructing A, in culture early stage, it can be seen that colony is still limited to micropore bottom.Continue in culture After growth is amounted to 14 days, colony has been grown beyond micropore and cell is occurred in around the hole containing colony Most aperture in.The micropore obviously with this construction is not enough to accommodate colony after cultivating 14 days.Therefore, CFU-GM number during inoculation can not possibly be counted using this construction.

Referring now still to Figure 10, with regard to constructing B-E, in all cases, observe when the time point of early stage is cultivated It is restricted in each hole to cell, now colony still very little.When later time point is cultivated, it is seen that As micropore depth increases or the reduction of wall angle, the micropore of cell spreads to micropore and reduces.With maximum pore volume Construction (construction E) show that colony is restricted to completely in each hole, do not occur after 14 days in culture micro- Hole to micropore spreads.

Carry out extra experiment using microporous structure B-E, wherein by fluid medium from human cord blood Hemopoietic progenitor cell with 21-26 colony/cm²Density be seeded in the micropore of advance moistening and be passed to Gravitational settling is into micropore.The Colony forming number and colony for evaluating 7 or 14 days micropores after inoculation accommodates situation. For both cultures in 7 days and 14 days, as shown in following table, the colony number observed when wall angle increases increases Plus.

The exception of this trend is construction B in 7 days CFC are determined.During here is determined, deposit in all micropores In high cellular context, this makes it difficult to exactly count microcolony.This cellular context most-likely due to Overflow and spread to caused by adjacent cells with two-forty because wall angle is shallow.These data displays are for most Large scale depth ratio is 1.9: the micropore of 1-1.1: 1 scope, and wall angle is less than 30 degree, more specifically, wall angle is little It is in 20 degree, or desirable.

Evaluation of embodiment 4- to the cell culture system comprising sub-micro hole

Hematopoietic progenitor cells in fluid medium are seeded to and are contained within the thin of one group of sub-micro hole in each micropore In born of the same parents' culture dish.Micropore in cell culture system place at the top of micropore is foursquare, and with 1.0mm Pore widths, the micropore depth 105 of 1.0mm and 15 degree of wall angle.Sub-micro hole place is at the top of sub-micro hole It is foursquare, the pore widths with 0.37mm, the micropore depth of 0.5mm and 15 degree of wall angle.

With about 7 colony/cm²Colony density inoculating cell culture apparatuses and by slow speed centrifugation by cell In being settled down to sub-micro hole.The cell culture system of inoculation subsequently 37 DEG C license culture environment and contain 5% CO₂Humidifying atmosphere in be incubated.The Colony forming of culture is observed by bright-field microscope after being incubated 7 days And it is imaged (Figure 11 A) using CCD digital cameras.Hole indicated by an arrow contains after being incubated 7 days at 37 DEG C There is the colony from hematopoietic stem cell ancestors.Subsequently use living cells mark MTT (brominations 3- (4,5- bis- Methylthiazol -2- bases) -2,5- diphenyltetrazolium bromides) staining cell with using CCD color scanners by macroscopic view point Colony is visualized (Figure 11 B) by analysis.In short, the staining is included in the culture medium containing MTT being incubated Culture is until observe that enough colors are formed.Then by removal culture medium (by liquid relief) and to sub-micro Add and remove suitable lavation buffer solution to wash the cell of dyeing in hole.Repeat this washing step until substantially Without background coloration.

It was found that being firmly fixed of colony is in sub-micro hole, and before or after dyeing and washing step Without the cell for freely floating.It was observed that can easily by the closely knit and discrete colony of microscopic counting, And obtain suitable colony count using microscope and macro approach.

Figure 11 A are displayed in the colony grown in sub-micro hole, and Figure 11 B show coloration result.Go out in this image Existing at least 6 positive 4 sub-micro holes in sub-micro hole and cluster, 4 sub-micro holes are in the upper right corner of image Flock together.Total colony frequency in based on this image, this group in 4 holes is most likely to be by single ancestral First produce, the single ancestors have the multiplication potentiality of height and grow beyond sub-micro hole.This enforcement Example clearly illustrates the advantage using sub-micro hole.

The dyeing of embodiment 5- colony

Containing with various constructions micropore Tissue Culture Dish in have rated be commonly used to biological sample and Three kinds of colouring methods of the dyeing of cell culture samples.The cell culture system tested in this embodiment is not wrapped Hole containing sub-micro.Methods described includes：(a) with enzyme alkali phosphatase (AP) be coupled for cell surface marker The antibody labeled cells of thing, then add naphthol phosphoric acid salt substrate and Fast-Red chromogens so as to cause redness Precipitate is produced, and (b) is made using MTT (bromination 3- (4,5- dimethylthiazole -2- bases) -2,5- diphenyltetrazolium bromides) Living cells for metabolism substrate are dyeed, and MTT changes into visible dyestuff by living cells, and (c) uses tissue Learn the unspecific staining of lining stain Evans blue (Evans Blue).For every kind of colouring method, containing collection The cleaning of the cell culture system for falling first passes through and removes culture medium (by liquid relief) and add suitable clear to micropore Wash buffer, then removes cleaning buffer solution after short incubation period.Weight is needed according to every kind of staining protocols Multiple this washing step.The result of dyeing shows in fig. 12.

Figure 12 A are the examples of the colony of the antibody staining being coupled using alkali phosphatase.These colonies are included in In cell culture system with micropore, micropore place at the top of micropore is foursquare, and pore widths are 0.8mm, wall angle is 15 degree, and micropore depth is 0.5mm.Colony is significantly dyed redness, background stainings It is minimum.In addition, the interference occurred to colony is not observed when staining procedure is received.Figure 12 B and 12C Display is included in the colony in the cell culture system with micropore, and micropore place at the top of micropore is square Shape, pore widths are 1.0mm openings, and wall angle is 15 degree, and micropore depth is 1.0mm.Colony is distinguished Dyeed with Evans blue and MTT.As antibody labeling method, appearance interference colony is not observed, and Colony is colored with the high contrast level with background.

Embodiment 6- to formed colony CFU-GM it is quantitative

The Colony forming culture to hemopoietic progenitor cell is carried out in cell culture system in liquid culture, while Standard Colony forming raji cell assay Raji is carried out to identical cell sample in semisolid culturemedium.In this embodiment The cell culture system of middle test does not include sub-micro hole.By the cell suspension inoculation in fluid medium extremely In cell culture system with micropore, micropore place at the top of micropore is foursquare, with 1.0mm Pore widths, the micropore depth of 1.0mm and 15 degree of wall angle.By slow speed centrifugation by cell settlement to micro- Kong Zhong.Will be in semisolid culturemedium (Methocult^TM, Stemcell Technologies) in same cell sample In the Tissue Culture Dish of the suspension inoculation of product to standard.In either case, culture dish is inoculated with about 5-15 colony/cm²Colony density, subsequently 37 DEG C license culture environment and contain 5%CO₂It is wet It is incubated in activating QI atmosphere.14 days post-evaluation Colony formings of culture and the total colony number of comparison.

Figure 13 illustrates the colony observed in the cell culture system comprising micropore and standard cell lines culture dish Dependency (being standardized as the colony number per 1000 cells being inoculated in culture dish) between number.Slope Be of about 1 significant correlation demonstrate comprising micropore cell culture system in colony determine with it is current Standard CFC determine good uniformity.The cell culture system for describing in this application thus provides one Plant the quantitative appropriate method of the CFU-GM to forming colony.

Embodiment 7-CFC setting-out line scope

The cell culture system comprising micropore is moistened in advance by slow speed centrifugation fluid medium.In this reality Apply the cell culture system tested in example and do not include sub-micro hole.The micropore tested in the present embodiment is on micropore top It is foursquare, the pore widths with 1mm, the micropore depth of 1mm and 15 degree of wall angle at portion.Will Liquid suspension from the hemopoietic progenitor cell of the luxuriant and rich with fragrance separable human cord blood of freezing is seeded to cell culture system In and be passed to gravity with about 7-57 colony/cm²Expected density sedimentation into micropore, cell concentration from 1x10⁴Individual cell/micropore is gradually increased to 1x10⁵Individual cell/micropore.License of the cell culture system at 37 DEG C Culture environment and contain 5%CO₂Humidifying atmosphere in be incubated.Cell culture system is evaluated after inoculation when 7 days Total colony number.

In fig. 14, positive hole (data point is shown as rhombus) number is not linearly increasing with cell implantation concentrations. Positive hole can be derived from single ancestors, or from more than one ancestors.Add to given micropore culture vessel Ancestors more (changing with the volume of ancestors' frequency, cell concentration and addition), each micropore is inoculated with more than one The probability of ancestors is higher.In the present embodiment, culture vessel has about 1000 micropores and therefore works as About 50% is positive when each ware bed board has highest cell number.Based on the frequency in the positive hole observed, Using Poisson distribution by determining expected ancestors' number needed for the positive number of perforations can be produced come to measure Output carry out linearisation.Figure 14 shown once apply this correction, the cell and ancestors' number of bed board it Between relation be linear (data point be shown as square).The number of correction represents the ancestors in initial sample Total number estimated value.

Magnetic Isolation of embodiment 8- to the CFU-GM of formation colony

With aqueous buffer solution (PBS containing 2%FBS) by the cell of the micropore with same configuration of embodiment 7 Culture apparatuses any air remaining in moistening to remove micropore.By phenanthrene can (Stemcell Technologies, 07907) mononuclear cell in density gradient centrifugation enrichment human cord blood sample is simultaneously resuspended in cell above-mentioned slow In rushing liquid.By this cell suspension and the magnetic particle (Stemcell Technologies, D- microgranule) of glucosan coating It is mixed with anti-glucosan/anti-CD34 mixtures of antibodies (Stemcell Technologies, CD34+ select mixture) Close.After hemopoietic progenitor cell in incubation is to allow suspension specifically binds with magnetic microcarrier, by mixture Add to cell culture system.Then cell culture system is placed on flat Magnet (LifeSep 384F).See Migrate downward into along magnetic field gradient to microcarrier and be settled down in micropore.Observe in the period of less than 2 minutes Suspension becomes limpid, while seeing that dark-coloured precipitate is formed in microvia bottom.Remove excessive upper by liquid relief Clear liquid is simultaneously changed with the fluid medium containing cytokine so that proliferation of hematopoietic progenitors.In same configuration Other culture in cell culture system and in semisolid culturemedium is such as implemented above as control inoculation Described in example 6.Cell culture system and to impinge upon licensing environment (37 DEG C, 5%CO₂, humidified incubator) and middle incubation The period of 7 days and the formation of observation port inner cell colony.

Bright-field microscope observation result discloses uniform point of microcarrier bead in the micropore of cell culture system Cloth.However, in each micropore, it is found that pearl corresponds to the direction (Figure 15 A) of magnetic field gradient towards micropore Edge aggregation.Although after inoculation magnetic bead causes each cell smudgy, after culture 7 days, can Colony (Figure 15 B) has been respectively formed in observe each hole in whole cell culture system.Use wherein Total colony count in the cell culture system of magnetic microcarrier selectivity sedimentation hemopoietic progenitor cell with compareing carefully Observe in born of the same parents' culture apparatuses and the culture that carries out in semisolid culturemedium in petri diss (Petri dishes) The colony count for arriving is quite (Figure 16).This is illustrated in the cell culture system comprising micropore, by using magnetic Property microcarrier and the mixtures of antibodies special to the unique markers on cell surface, by by required cell Selectivity is settled down in micropore, can be carried out the quantitative colony to particular cell types and be determined.

Claims

1. a kind of cell culture system, comprising：

The hole limited by least one hole side wall and bottom hole wall；

Multiple micropores in the hole, each micropore from the bottom hole wall by upwardly extending and by pore fluid body It is described micro- in the long-pending at least one microporous side wall separated with the pore fluid volume of adjacent cells and the hole The first common fluid volume defining above hole, each micropore includes micropore top and micropore bottom, and Microporous side wall between wherein adjacent micropore converges to form summit；With

The second public stream in one group of sub-micro hole and each micropore in each micropore above described group of sub-micro hole Body volume, one group of sub-micro hole is used to accommodate one or more cells in each sub-micro hole, and each is sub- Micropore includes sub-micro hole top, sub-micro bottom hole portion and upwardly extending by sub- pore fluid from the bottom hole wall Volume and at least one sub- microporous side wall that the sub-micro hole fluid volume in adjacent sub-micro hole separates.

2. the cell culture system described in claim 1, wherein each sub-micro hole is further by the microporous side wall A side wall a part restriction.

3. the cell culture system described in claim 1, wherein the bottom hole wall is transparent or translucent.

4. the cell culture system described in claim 1, wherein every group of sub-micro hole includes 4 arranged with 2x2 arrays Individual sub-micro hole.

5. the cell culture system described in claim 1, wherein every group of sub-micro hole include with 2x1,3x1,3x2, Multiple sub-micro holes of the array arrangement of 3x3 or bigger.

6. the cell culture system described in claim 1, wherein each sub-micro hole are on cross-sectional area from the sub-micro Hole top is gradually reduced to the sub-micro bottom hole portion, and optionally, wherein each sub-micro hole is frustoconical Or frustum.

7. the cell culture system described in claim 1, wherein each micropore are on cross-sectional area from the micropore top Portion to the micropore bottom tapers into, and optionally wherein each micropore is frustoconical or frustum 's.

8. the cell culture system described in claim 1, wherein the sub-micro hole, micropore and hole are integrally formed.

9. the cell culture system described in claim 1, further comprising the magnetic being positioned at below the sub-micro hole Or magnetizable element.

10. the cell culture system described in claim 9, wherein the magnetizable element is to be embedded in the bottom hole wall Interior wiregrating.

Cell culture system described in 11. claim 1, wherein micropore top has at least 100 microns of width The depth of degree and/or each micropore between the top and the bottom is at least 75 microns.

Cell culture system described in 12. claim 1, wherein each micropore are included at the micropore top most Micropore depth between large scale and micropore top and the micropore bottom, and the full-size It is 1.1 with the ratio of the micropore depth:1 to 1.9:1.

Cell culture system described in 13. claim 12, wherein the full-size is at least 140 microns.

Cell culture system described in 14. claim 12, wherein the micropore depth is at least 75 microns.

Cell culture system described in 15. claim 1, wherein angle of the microporous side wall relative to vertical direction It is optionally less than 20 degree less than 30 degree.