CN107058098A - A kind of device and method of the cell patterning culture based on 3D printing technique - Google Patents
A kind of device and method of the cell patterning culture based on 3D printing technique Download PDFInfo
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Abstract
The invention belongs to 3D printing technique and technical field of cell culture, in particular it relates to the device and method that a kind of cell patterning that can be quickly prepared based on 3D printing is co-cultured.This application provides a kind of 3D printing can be utilized quickly to prepare the micro element technology co-cultured for cell culture and cell patterning, and the device can be easily compatible with existing cellular biological technique, such as micro-imaging, cellular immunity dyeing etc..The patterning to various kinds of cell can be conveniently realized, and microscopic image analysis and immunohistochemical analysis can be carried out to cell, the result of quantification is obtained.The co-cultivation that can more rapidly conveniently realize various kinds of cell for scientific research personnel provides apparatus and method.
Description
Technical field
The invention belongs to 3D printing technique and technical field of cell culture, in particular it relates to which one kind can be with 3D
The device and method that the cell patterning quickly prepared based on printing is co-cultured.
Background technology
The patterning main purpose of cell is to make cell growth in region set in advance, forms specific pattern.Micro- pattern
Change technology can control the spatial distribution of cell under micro-meter scale, therefore to organizational project, biosensor technology, Yi Jiji
The research of plinth biological question is significant.Micro-patterning technology can to including population of cells's size, ecotone away from
Direct regulation and control is carried out from the interaction between, of the same race or heterogenous cell, and the control for these spatial parameters can direct shadow
Ring metabolism and the cell function of cell.For example, the size of human umbilical vein endothelial cell and occupied space can be directly affected
It, which breaks up, tends to, and the interaction between macrophage, fibroblast and endothelial cell can promote the blood vessel of endothelial cell to give birth to
Into trend.Therefore, the micro-patterning technology of cell is clearly a kind of strong biomedicine for studying cell behavior and function
Engineering tools.
In the past ten years, cell patterning techniques have been widely used in research cell-cell interaction, cell
Other functions of response, polarization growth, cell differentiation and cell to local environment.In general, cell is patterned
Method can be classified as two major classes：One class is to use different chemical reagent, such as self assembled monolayer and people's fibrinoid,
The surface of culture carrier is modified, repels it or promotes the attaching of cell；Another kind of is cell using physical method
Specific region is fixed on, pipeline, Wei Keng, cut-off and the small hook for typically thering is micro Process to be made.There are some reports by both sides
Method dexterously combines the intercellular interaction of research.
But, realize that the process of cell patterning is generally complex currently with physical method, such as photoetching skill
Art and soft lithography, its preparation facilities process is more complicated, it is necessary to which specific instrument and equipment, common laboratory is difficult to realize.With
The gradually development and progress of 3D printing technique, its can realize it is multiple dimensioned under manufacture.3D printing, also known as increasing material manufacturing, belong to
One kind of RP technique, it is the skill that almost any shape of 3D solid is manufactured based on a kind of mathematical model file
Art.3D printing with powdery metal or plastics etc. can jointing material, come constructed object by way of successively stacking accumulation, i.e.,
" layer appearance method ".3D printing is different from traditional mechanical manufacturing technology, and the latter is generally using cutting or drilling technique（Subtract material work
Skill）Realize.Past, it was often used for modeling in fields such as Making mold, industrial designs, was now increasingly used for some products
Direct manufacture.Particularly some high value applications have had the parts printed using this technology, it is meant that " 3D is beaten
The popularization of this technology of print ".3D printer on the market has been carried out the manufacture of micro-meter scale now, and precision is also carried year by year
It is high.Concentrated it is worth noting that 3D printing technique is studied in the application of biomedical engineering field also in the exploratory stage, a lot
In terms of to the reparation of support and sclerotin joint etc., and the application in terms of cell biology, organizational project is actually rare.
The device of micron accuracies is manufactured using 3D printing and applies it in the micro- pattern of cell biology particularly cell
Invention in change yet there are no report.
The content of the invention
In view of the above-mentioned problems, this application provides one kind 3D printing can be utilized quickly to prepare for cell culture and cell
The micro element technology co-cultured is patterned, and the device can be easily compatible with existing cellular biological technique, it is such as aobvious
Micro- imaging, cellular immunity dyeing etc..Can conveniently realize the patterning to various kinds of cell, and cell can be carried out it is micro- into
As analysis and immunohistochemical analysis, the result of quantification is obtained.Various kinds of cell can be more rapidly conveniently realized for scientific research personnel
Co-cultivation provide apparatus and method.
The present invention is achieved by the following technical solutions：
A kind of device of many cells patterning culture based on 3D printing technique, it is characterised in that
Described device includes multigroup hollow tube being arranged concentrically, and every group of hollow tube includes an outer hollow tube and at least one
Individual interior hollow tube, the bottom of interior hollow tube and the bottom of outer hollow tube are concentric；Described device uses 3D printing technique system
Standby, the bottom of hollow tube is arranged on cell culture utensil, the extensible cell culture utensil in bottom of interior hollow tube；It is interior
The external diameter of hollow tube is less than the internal diameter of outer hollow tube.Between interior hollow tube, outer hollow tube and interior hollow tube
White space inserts different types of cell respectively, and interior hollow tube is removed after cell is adherent, various kinds of cell is patterned phase
Neighbour, while the wall thickness of interior hollow tube causes different cell compartments to retain certain interval, this device can be more for convenient observation
Intercellular migration and interaction.
Further, described device includes upper and bottom section, and bottom point includes multiple outer hollow tubes and lower support
Portion；Upper part includes multiple interior hollow tubes and upper supporting part, and interior hollow tube is connected with upper supporting part, upper supporting part and lower branch
Support part is removably mutually matched, upper supporting part and lower supporting part cause when coordinating each interior hollow tube respectively with one it is outer in
Blank pipe body bottom is with centrally disposed；The drawable cell in bottom of interior hollow tube is may be such that when upper supporting part and the dismounting of lower supporting part
Cultivate utensil.
Further, the upper-end inner diameter of interior hollow tube is more than the bottom internal diameter of interior hollow tube.
Further, the internal diameter of hollow tube is from micron order to Centimeter Level.
Further, by that two supporting part interpolations will combine up and down, reversible sealing-in can be realized.
Further, the raw material of 3D printing have good cell compatibility and environment friendly.
A kind of method of many cells patterning culture based on 3D printing technique, it is characterised in that methods described is using power
Profit requires 1 described device, and the described method comprises the following steps：It is prepared by device：Many cells pattern is prepared using 3D printing technique
Change culture apparatus, device is placed on cell culture utensil；
Add the aaerosol solution of cell：By the aaerosol solution of cell be injected separately into interior hollow tube, interior hollow tube with it is outer hollow
Between body, put it into cell culture incubator and cultivate；The mlCell suspended solution of injection is identical in different hollow tubes or not phase
Together；
Hollow tube in removing：After cell attachment, interior hollow tube is vertically removed, and changes fresh culture medium and continues to cultivate,
Observe the migration and interaction of cell.
Further, using the material that one layer of promotion cell attachment is incubated on preceding cell culture utensil.
Further, in the aaerosol solution step of addition cell, the aaerosol solution of cell is fibroblastic suspension
With the suspension of human desmocyte sarcoma cell, cell concentration equal 105Individual/ml, the temperature of cell culture incubator is 37 DEG C, carbon dioxide
Volumetric concentration is 5%, and incubation time is 4-6 hours.
Further, cell culture utensil is Tissue Culture Dish or culture plate, and the substrate of cell culture utensil is glass, silicon
Piece, metal or high polymer material.
The advantageous effects of the present invention：
Limitation of the conventional apparatus chip preparation for complex technology such as photoetching technique has been broken in the application of 3D printing technique, any
Laboratory can easily be realized, reduce the requirement to laboratory clean room and complex device.And can be according to experiment
As a result change optimization design real-time, without the die sinking plate again as photoetching again, it is to avoid the waste of time and money.More
Importantly, it can quickly design a large amount of different models or structure according to the experiment of oneself, to reality for photoetching
The technical requirements of personnel are tested than relatively low, while improving operating efficiency, experiment purpose can be also reached, can be straight using the present invention
The relative movement for observing various kinds of cell seen and interaction.
Brief description of the drawings
Fig. 1 is the schematic diagram of 3D printing device；
Fig. 2 is that diverse location pours into different cells in 3D printing device；
Fig. 3 is to remove the cell pattern schematic diagram formed after superstructure；
Fig. 4 is Fig. 3 enlarged drawings of one of them；
Fig. 5 is the delay figure of different pharmaceutical cell migration influence；
Fig. 6 is the schematic cross-section of 3D printing device;
In figure：A. part under device, part on B. devices, hollow tube bottom internal diameter, b. in C. cell culture utensils substrate, a.
Hollow pipe outer wall and outer hollow inside pipe wall spacing are from hollow tube pipe thickness, e. outside, d. in interior hollow tube pipe thickness, c.
Interior hollow tube upper-end inner diameter, h1Interior hollow tube height, h2Outer hollow tube height.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is explained in further detail.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, and
It is not used in the restriction present invention.
On the contrary, the present invention covers any replacement done in the spirit and scope of the present invention being defined by the claims, repaiied
Change, equivalent method and scheme.Further, in order that the public has a better understanding to the present invention, below to the thin of the present invention
It is detailed to describe some specific detail sections in section description.Part without these details for a person skilled in the art
Description can also understand the present invention completely.
Embodiment 1
As shown in Figure 1 there is provided the device that a kind of many cells are co-cultured, including：The device and Tissue Culture Dish of 3D printing or training
Support plate substrate；Graphics required for being designed by Three-dimensional Design Software produces corresponding model via 3D printer and is used as
Experimental provision, the present apparatus is mainly based on tubular structure, and internal diameter can be realized from micron order to Centimeter Level；The 3D printing model
It is arranged in the Tissue Culture Dish or culture plate substrate；The substrate surface of Tissue Culture Dish or the culture plate substrate, which is not done, to be located
Reason, or it is incubated the material that one layer of Collagen type-I etc. promotes cell attachment.Its manufacturing process is without complicated photoetching technique, simple side
Just, it is easy to operate.
Fig. 1 is the schematic diagram of 3D printing device, and it is mainly made up of two parts, including upper and bottom section, lower part
Including nine connected a diameter of 1cm, a height of 1mm outer hollow tube and lower supporting part；Upper part includes multiple interior hollow tubes
Body and upper supporting part, interior hollow tube are connected with upper supporting part, and upper supporting part and lower supporting part are removably mutually matched, upper branch
Support part causes each interior hollow tube respectively with an outer hollow tube bottom with centrally disposed when coordinating with lower supporting part；Upper branch
The drawable cell culture utensil in bottom of interior hollow tube is may be such that when support part and the dismounting of lower supporting part.
The present embodiment lower middle portion includes nine connected outer hollow tubes, and positioned at the one of nine outer hollow tube surroundings
Framework, the both sides of framework set fluted, and framework is used as lower supporting part.
Upper part includes interior hollow tube and " door " shape framework, and " door " shape framework includes a horizon bar and two vertical
Bar, vertical bar and horizon bar are vertical.The upper end of interior hollow tube is arranged on horizon bar, and horizon bar relevant position is provided with hole,
The hollow hole of the hole and interior hollow tube is communicated." door " shape framework is used as upper supporting part.
The horizon bar of the groove of framework and " door " shape framework is adapted, and horizon bar insertion groove can be realized into internal hollow tube
The fixation and support of body, now the bottom of interior hollow tube and the bottom of an outer hollow tube are arranged concentrically.
Originally it is every three outer row of hollow tube one in strength, nine connected outer hollow tubes are divided into three rows, each bottom
Part in three identicals need to be had by dividing, but the application is not limited to the setting.
The bottom internal diameter and upper-end inner diameter of interior hollow tube are differed, and upper-end inner diameter is more than bottom internal diameter, and convenient injection is thin
Cytosol.
The horizontal interface of hollow tube is circle in the present embodiment, but the invention is not restricted to circle, interface can also be it is square,
Prismatic or other irregular shapes.
Fig. 6 is the Longitudinal cross section schematic of 3D printing device, designates the size of key position.
Wherein, a is the diameter in initial circular region shared by inner ring cell；B is inside and outside two circles iuntercellular blank circle ring area
Ring width, show as the pipe thickness of interior hollow tubular body；C is the ring width of initial circle ring area shared by the cell of outer ring；D is shown as
The pipe thickness of outer hollow tubular body；E shows as the groove inner width of interior hollow tubular body connection, and it influences inner ring cell suspension
Volume；h1Show as the outer height of interior hollow tubular body, the volume of the inside and outside two circles cell suspension of influence；h2Show as outer hollow tube
The height of shape body, influences the volume of outer ring cell suspension.Specific size is as follows：A is 800 μm, and b is 1000 μm, and c is 3600 μm,
D is 1000 μm, and e is 4000 μm, h1For 2000 μm, h2For 1000 μm.
The substrate of Tissue Culture Dish or culture plate is glass, silicon chip, metal or high polymer material such as polystyrene, it is easy to
Obtain, it is cheap.
3D printing device shape size flexibility and changeability, size can be realized from micron order to Centimeter Level.
3D printing device can realize reversible sealing-in by that two supporting part interpolations will combine up and down.
The raw material of 3D printing be degradable PLA, ABS engineering plastics or other can be used for 3D printing macromolecule
Material etc. has good cell compatibility and environment friendly.
To achieve the above object, present invention also offers a kind of method that many cells are co-cultured, using said apparatus, specifically
Comprise the following steps：
Step 1: preparing the Tissue Culture Dish or culture plate substrate, the 3D printing device is prepared, it is mainly by two parts structure
Into bottom is nine connected a diameter of 1cm, a height of 1mm tubular body, as shown in structure A in Fig. 1；It is concentric with its to be
A diameter of 800 μm of tubular body, as shown in structure B in Fig. 1, specific size reference picture 6；
Step 2: being made into the Collagen type-I that concentration is 300 μ g/ml in water and glacial acetic acid solvent, grasped under 4 DEG C of cryogenic conditions
Make, the Collagen type-I is added dropwise in the culture dish or culture plate substrate, is put in incubator and PBS is used after 12h
（1X）Solution slowly cleaning 3 times, are placed in and dry naturally at room temperature, the 3D printing device is placed on and has been incubated one layer of rat-tail
In the Tissue Culture Dish or culture plate substrate of collagen, it is therefore an objective to make cell adherent under truth in closer organism
Growth, by that two supporting part interpolations will combine up and down, can form reversible sealing；
Step 3: as shown in Fig. 2 preparing the aaerosol solution of cell and injecting the Tissue Culture Dish for being incubated one layer of Collagen type-I
Or in the device of culture plate substrate, put it into cell culture incubator and cultivate cell 4-6 hours.Fig. 2 is in 3D printing structure
Diverse location pours into different cells, and bottom dark colored portion is that light-colored part is behaved into fibroblast suspension, upper grooves
Fibrosarcoma cell suspension, will not occur cross pollution between the two.
Step 4: after cell attachment, it is vertical to remove interior hollow tube, and change fresh culture medium continuation culture.Its dozen
The limitation to conventional lithographic techniques has been broken, a kind of method that many cells based on 3D printing technique are co-cultured is established.Fig. 3
It is to remove the cell pattern schematic diagram formed after superstructure, middle light-colored part behaviour fibrosarcoma cells, outside is deep
Color part is fibroblast, and white space is the part of cell co-migrate between the two.
It can be needed to inject different cell solutions, the present embodiment note between inside hollow tube and outer hollow tube according to practical study
What is entered is human desmocyte sarcoma cell and fibroblast, but not limited to this.
It can also be used to study effect and function of the different pharmaceutical under cell interaction, as shown in figure 5, having obtained not
Co-cultivation figure with the conditions of, is that subsequent biological analysis provides the foundation.Fig. 5 is prolonging for different pharmaceutical cell migration influence
Shi Tu.Middle light-colored part behaviour fibrosarcoma cells, outside dark parts are fibroblast.The first row be control group not
Dosing thing, the second row is the experimental group containing Cytochalsin D, and the third line is the experimental group containing GM6001.Can from figure
To find out the migration of Drug inhibition cell.
The aaerosol solution of cell in step 3 is the suspension of fibroblastic suspension and human desmocyte sarcoma cell
Liquid, cell concentration equal 105Individual/ml, the temperature of the cell culture incubator is 37 DEG C, carbon dioxide volumetric concentration is 5%, during culture
Between be 4-6 hours.
The suspension of cell is drawn in step 3 with 1ml syringes respectively, as shown in Fig. 2 fibroblastic cell suspension
Blank position between two kinds of tubular bodies is injected into, the suspension of human desmocyte sarcoma cell is injected into the recessed of the interior hollow tubular body of connection
In groove.
Step 4, as shown in figure 4, i.e. after cell attachment, removing interior hollow tubular body, human desmocyte sarcoma can be observed thin
Born of the same parents are only grown in the range of a diameter of 800 μm or so of pattern center, and its outer ring is 1mm or so blank circle ring area, outermost layer
For fibroblast, wash non-attached cell off 3 times with PBS, change fresh culture into, place into the cell culture incubator
In, the migration and interaction of two kinds of cells are observed, so we change by calculating human desmocyte sarcoma cell occupied area
Migration and variation of the cancer cell under fibroblastic effect can be obtained.
Because using 3D printing technique, the internal diameter of hollow tube arrives greatly the small micron order that arrives of Centimeter Level and can be achieved, and is not limited to this
Embodiment numeral.
Embodiment 2
The present embodiment is substantially the same manner as Example 1, and difference is, device prepared by 3D printing technique includes 2 interior hollow tubes.
Claims (10)
1. a kind of device of many cells patterning culture based on 3D printing technique, it is characterised in that described device includes multigroup
The hollow tube being arranged concentrically, every group of hollow tube includes an outer hollow tube and at least one interior hollow tube, interior hollow
The bottom of body and the bottom of outer hollow tube are concentric；Described device is prepared using 3D printing technique, and the bottom of hollow tube is equal
It is arranged on cell culture utensil, the extensible cell culture utensil in bottom of interior hollow tube；The external diameter of interior hollow tube is less than
The internal diameter of outer hollow tube.
2. device as claimed in claim 1, it is characterised in that described device includes upper and bottom section, bottom point includes many
Individual outer hollow tube and lower supporting part；Upper part includes multiple interior hollow tubes and upper supporting part, interior hollow tube and upper support
Portion is connected, and upper supporting part and lower supporting part are removably mutually matched, and upper supporting part causes when coordinating with lower supporting part in each
Hollow tube is respectively with an outer hollow tube bottom with centrally disposed；Upper supporting part and lower supporting part dismounting when may be such that it is interior in
In the extensible cell culture utensil in bottom of blank pipe body.
3. device as claimed in claim 1 or 2, it is characterised in that the upper-end inner diameter of interior hollow tube is more than interior hollow tube
Bottom internal diameter.
4. device as claimed in claim 1 or 2, it is characterised in that the internal diameter of hollow tube is from 1*10-6~1*10-2Rice.
5. device as claimed in claim 2, it is characterised in that by that two supporting part interpolations will combine up and down, can realize reversible
Sealing-in.
6. device as claimed in claim 1 or 2, it is characterised in that the raw material of 3D printing have good cell compatibility and
Environment friendly.
7. a kind of method of many cells patterning culture based on 3D printing technique, it is characterised in that methods described uses right
It is required that 1 described device, and the described method comprises the following steps：It is prepared by device：Many cells patterning is prepared using 3D printing technique
Culture apparatus, device is placed on cell culture utensil；
Add the aaerosol solution of cell：By the aaerosol solution of cell be injected separately into interior hollow tube, interior hollow tube with it is outer hollow
Between body, put it into cell culture incubator and cultivate；The mlCell suspended solution of injection is identical in different hollow tubes or not phase
Together；
Hollow tube in removing：After cell attachment, interior hollow tube is vertically removed, and changes fresh culture medium and continues to cultivate,
Observe the migration and interaction of cell.
8. method as claimed in claim 7, it is characterised in that promote cell attachment using being incubated one layer on preceding cell culture utensil
Material.
9. method as claimed in claim 7, it is characterised in that in the aaerosol solution step of addition cell, the aaerosol solution of cell
For fibroblastic suspension and the suspension of human desmocyte sarcoma cell, cell concentration equal 105Individual/ml, cell culture incubator
Temperature be 37 DEG C, carbon dioxide volumetric concentration be 5%, incubation time is 4-6 hours.
10. method as claimed in claim 7, it is characterised in that cell culture utensil is Tissue Culture Dish or culture plate, cell training
The substrate for supporting utensil is glass, silicon chip, metal or high polymer material.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108823165A (en) * | 2018-04-17 | 2018-11-16 | 广州波奇亚标准及检测技术有限公司 | It is a kind of to change the new method that surface wettability realizes three-dimensional cell cultivation based on laser |
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| US11993767B2 (en) | 2019-11-27 | 2024-05-28 | Cellbricks Gmbh | Method for producing 3D, biocompatible polymer scaffold with a cell-filled cavity |
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