CN103468635A - Three-dimensional cell scaffold printing method based on cell state feedback - Google Patents
Three-dimensional cell scaffold printing method based on cell state feedback Download PDFInfo
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Abstract
The invention discloses a three-dimensional cell scaffold printing method based on cell state feedback. According to the invention, a marker is added during cell sap culture, so that the purpose of real-time tracking of cell distribution is realized; in a process of manufacturing a support material, test solutions for detecting cell secretion are mixed, so that the purpose of real-time detection of a differentiation process of stem cells is realized. Through the cell tracking and the real-time detection of the cell differentiation process, real-time monitoring of sedimentation of gelatin carrying cells is realized, so that the study of a direct relation between cell survival and differentiation and all parameters (such as deposition velocity and piezoelectric pulse width) of the sedimentation technology is facilitated, thereby greatly shortening the study period and reducing the cost.
Description
Technical field
The present invention relates to the cell printing field, especially relate to a kind of three-dimensional cell support Method of printing based on the cell state feedback.
Background technology
Rapid shaping (RP) technology is the advanced manufacturing technology that the nineties grows up, under the control of computer, the RP system can be according to the shape of part, a cross section with certain small thickness and specified shape of each making, and then they are successively bondd, just obtained the part of the solid of required manufacture.The RP system shaped material difference used that different company manufactures, the principle of work of system is also different, but its ultimate principle is all the same, that is exactly " layering manufacture, successively stack ".Utilize rapid shaping (RP) technology, can in the situation that, without preparing any mould, cutter and tooling fixture, directly accept product design (CAD) data, produce fast exemplar, mould or the model of product innovation.
At bioengineering field, rapid shaping technique is to manufacture a good technical tool of support (three-dimensional cell support).These technology based on model can, by carry out successively deposition of material on worktable, obtain porous 3D support.In the process of structure support, every one deck is all to be deposited as the fibre network (material and space) that can interpenetrate.Can form like this one has porous and there is no the three-dimensional rack be connected.A controlled space support that has can well provide oxygen, nutriment to guarantee tissue culture to cell and tissue.The inside and outside structure of support can be drawn and be formed by CAD or CAM.Can produce replaceable healthy tissues and meet the 3D structure of its mechanical property by these modes.In addition, utilize computer tomography and Magnetic resonance imaging can obtain the support of histological structure.The diversity of this production support can more be conducive to study the regeneration situation of cell in the different tissues support.In the recent period, utilize the rapid shaping manufacture can provide the three-dimensional structure of Growth of Cells can create, its target is to manufacture equivalent viable cell.This technology, have a lot of people to be referred to as tissue or cell printing, also may be defined as many cells group deposition technique, is expected to imitate viable cell and arranged.Ultimate principle is a method that can more approach true cell, matrix and biomolecules tissue of design, and this method can help to disclose cell and cell, cell and matrix relation, and also can offer help for the cell regeneration in function transplanting.
The 3D deposition technique reaches its maturity to simple polymer support printing technique at present, and satisfactory organism support of simple printing has not been the Main Bottleneck of cell printing.Even can be for the gel stent difficulty of viable cell existence normal differentiation but can print one, polymer deposition support main drawback commonly used has the making of (1) cell and support asynchronous at present.The main method of modern cell deposition is first to be manufactured with the organic polymer support, inject viable cell again in support, its advantage is to guarantee to inject the viable cell surviving rate, but shortcoming is to control composition and the ratio of cell in support, the content that causes cell in support is because the restriction of supporting structure distributes inequality; (2) cell survives and real-time is lost in differentiation, and due to the Real-Time Monitoring that can't in injection process, realize cell, whether cell only survives after cell injects fully and cultivates for some time, could detect cell and whether survive the differentiation situation with cell.And do not meet while surviving condition when the made support, need to again make support and repeated experiments, this just causes really making a satisfactory support will consume a large amount of time energy.(3) surviving rate detects as detecting after deposition, when cell is injected in the process of support, the process death that cell may inject, this is owing to being injected into and arriving at cell in the time period in support predetermined position, cell can not obtain enough nutrients, and during this period, cell will be subject to extruding in various degree.In the impact of many extraneous factors, probably cause cell dead in advance, such result can not prove that the structural formula of support itself has problem, can produce deviation to experimental result.Experimental result produces the deviation meeting and then causes how invalid repeating to make the support process.
Summary of the invention
The invention provides a kind of three-dimensional cell support Method of printing based on the cell state feedback, in real time cell survival status, ratio and cytodifferentiation situation in support are detected to feedback in making processes, adjust three-dimensional cell support print parameters according to detecting data, and then obtain accurate three-dimensional cell support.
For solving the problems of the technologies described above, technical scheme provided by the invention is as follows:
A kind of three-dimensional cell support Method of printing based on the cell state feedback comprises:
(1) culture dish that stem cell is placed in to vital dye is cultivated dyeing, until, after the stem cell variable color, coloured stem cell is separated with colourless stem cell, takes out coloured stem cell;
(2) the coloured stem cell after dyeing is placed in to gel stoste and cultivates, after having cultivated, add the gel body lotion, filter the stem cell that obtains the gel parcel;
(3) stem cell that step (2) is obtained to gel parcel is packed in the syringe of 3 D-printing device, carry out the printing of three-dimensional cell support, in print procedure, read in real time the distributed intelligence of coloured stem cell on the three-dimensional cell support according to the distribution situation of coloured stem cell, final curing obtains the three-dimensional cell support;
(4) the three-dimensional cell stentplacement that obtains, after 20-48 hour, is immersed in stem cell initial stage differentiation detection reagent, detects stem cell initial stage differentiation information on the three-dimensional cell support;
(5), by the three-dimensional cell stentplacement 10-15 days obtained, utilize stem cell later stage differentiation information on Protein Detection equipment Inspection three-dimensional cell support;
(6) finally make the following judgment:
Meet the requirements if prepare the three-dimensional cell support, complete print procedure;
If it is undesirable to prepare the three-dimensional cell support, on the three-dimensional cell support that on the three-dimensional cell support that the distributed intelligence of coloured stem cell on the three-dimensional cell support step (3) obtained, step (4) obtain, stem cell initial stage differentiation information and step (5) obtain, stem cell later stage differentiation information is as feedback information, 3 D-printing device print parameters in set-up procedure (3), repeating step (1)-(6).
Below several preferred versions of technique scheme is described further:
Stem cell used in the present invention, can select various stem cells, such as adopting bone stem cell, hemopoietic stem cell etc.Three-dimensional cell support Method of printing in the present invention comprises cell cultures part and organism rack making part: the organism support is generally selected the gel-like organism, as the effective organic materials of anti-ultraviolet radiation and electromagnetic effect such as marine alga salt, except organism, holder part also should contain the proteolytic enzyme that noble cells is corresponding and detect test solution, as the soda acids such as phenolphthalein detect solution.
In step (1), in described cultivation dyeing course, adoptable dyestuff is for can mark surviving the vital dye of stem cell, common vital dye can be selected the normal saline solution of guinea green B or toluylene red, the concentration of vital dye can be determined according to actual needs, and the vital dye excessive concentration can cause the viscosity of stem cell excessive, the gel finally formed easily stops up the printing head of type printer, is unfavorable for follow-up printing; Concentration is too low, and Color is not good; As preferably, the weight percent concentration of described guinea green B or toluylene red is 0.01-0.05%.
In step (1), described coloured stem cell and colourless stem cell lock out operation can be selected by centrifugation; With respect to viable cell, dead cell is due to dehydration, the density relative reduce, and most of dead cell can swim in the normal saline solution of vital dye, by centrifugal operation, can realize fast separating of viable cell and dead cell, improves separation efficiency and disintegrate-quality.
In step (2), described gel stoste is generally selected sodium alginate aqueous solution, in described sodium alginate aqueous solution, sodium alginate concentration can be adjusted according to actual needs, experimental results show that, when the excessive concentration of sodium alginate, form gel and easily cause printing head to stop up, be unfavorable for follow-up printing; When the concentration of sodium alginate is too low, because gel strength is rarer, print lines and easily produce diffusion, reduced printing precision; As preferably, in described sodium alginate aqueous solution, the weight percent concentration of sodium alginate is 2-5%, and while selecting this concentration, the easier moulding of gel, improved whole printing effect and print quality.In addition, because ion can promote solidifying of gel sodium alginate, so, when configuration gel stoste, occur to solidify for avoiding gel stoste, as preferably, during the configuration sodium alginate aqueous solution, adopt deionized water.
In step (2), described gel body lotion is generally selected CaCl
2the aqueous solution, described CaCl
2the concentration of the aqueous solution can be adjusted according to actual needs, experiment showed, CaCl
2the concentration of the aqueous solution also is difficult for too high too low, CaCl
2the excessive concentration of the aqueous solution and too lowly all be unfavorable for follow-up printing.As preferably, described CaCl
2caCl in the aqueous solution
2mass percent concentration be 2-5%.
In step (2), coloured stem cell is placed in the time that gel stoste cultivates and can determines according to actual needs, the coloured stem cell of general warranty fully adapts to the gel environment, reduce the follow-up mortality ratio of stem cell, as preferably, it is more than 4 hours that described coloured stem cell is placed in the time that gel stoste cultivates.Coloured stem cell is long at gel stoste incubation time, can cause because nutriment is not enough dead, so as further preferably, it is 4～10 hours that described coloured corpuscle is placed in the time that gel stoste cultivates.In this step, after adding the gel body lotion, gel carries out Procuring, and Procuring time general control is at 0.5-3 minute, and the Procuring time is oversize, can make the formation gel viscosity excessive, is unfavorable for follow-up print job.
In step (3), described 3 D-printing device can be selected existing structure, such as selecting piezoelectric three dimension type printer etc.Design the structure of three-dimensional cell support and the movement locus of printing head by CAD and CAM Software on Drawing in this step, by the x of printing head and printing receiving plane, y, z tri-axles move the deposition injection and obtain any definite three-dimensional rack structure.In this step, curing operation generally selects UV-irradiation to solidify, and utilizes the reversable light sensitive characteristic by three-dimensional cell support solidifying and setting.The ultra violet lamp time is unsuitable long, and time of ultraviolet irradiation is long, can cause necrocytosis, for meeting solidifying requirements, as preferably, described time of ultraviolet irradiation is 1-3 minute, the general weak UV-light of selecting of ultraviolet light intensity gets final product, and for example can adopt the ultraviolet ray for sterilization of conventional medical science.In print procedure, the distribution situation of coloured stem cell has been determined the distribution situation that becomes viable cell, and wherein, coloured stem cell is the stem cell survived, and coloured moiety not, shows that cell is dead or there is no a stem cell.Thereby determine the content of different positions stem cell alive in support by determining that coloured stem cell distributes.
What in earlier stage secrete due to stem cell is alkali formula Phosphoric acid esterase, and the collagen protein of later stage secretion, so stem cell early stage and later stage differentiation situation need to adopt different detection meanss.In step (4), described stem cell initial stage differentiation detection reagent can be selected phenolphthalein indicator or pistil solution.Described phenolphthalein indicator is 0.5% phenolphthalein ethanolic soln.Before the three-dimensional cell support that printing is obtained is inserted cell initial stage differentiation detection reagent, for guaranteeing the bulk strength of three-dimensional cell support, generally need to place 20-48 hour, as preferably, can select to place 24 hours.In test process, utilize the phenolphthalein solution of preparation to observe the variation of color in the three-dimensional cell support, contain red positional representation phenolphthalein and redden, cell produces alkaline phosphatase, thereby detects the situation of initial cell differentiation, obtains stem cell initial stage differentiation information.In step (5), can detect situation and the position of cell later stage differentiation by Protein Detection equipment, obtain stem cell later stage differentiation information.Protein Detection equipment can be selected existing equipment.Before the Protein Detection equipment Inspection, as preferably, can be by the three-dimensional cell stentplacement that obtains 2 weeks.
In the process of rack making, by cell and support organism by a certain percentage mixed being incorporated in substratum cultivate, need to guarantee during cultivation that organic nutrient can enough make stem cell survive and break up.After determining ratio, by laminate structure, pack in syringe and utilize the piezo jet principle to be deposited.By CAD and CAM technology, design deposition three-dimensional arrangement and deposit track, move the three-dimensional cell support deposition that realizes loading cell by printing head and three axles of accepting plane.
In the process of actually operating, by the detection of fluorescent substance in stem cell or radioactive substance, can the ratio of Real Time Observation stem cell in the three-dimensional cell support, thereby by changing in real time the frequency of piezoelectricity, voltage, the factors such as pulse width are controlled the accounting of stem cell in organism.In the three-dimensional cell supporting structure formed, utilize different proteolytic enzyme and detection reagent to determine the differentiation situation of contained stem cell.For differentiated stem cells, corresponding enzyme detects the secretory product of specialized cell after differentiation and shows by detection reagent, thus the differentiation situation of real time reaction stem cell.
The present invention adopts and add marker when enchylema is cultivated, as dyeing solvent or the specific element of radioelement mark.Thereby reach the purpose of real-time follow-up cell distribution, in the process of making at timbering material, be mixed for being detected as the test solution of emiocytosis thing, reach the purpose of real-time detection differentiation of stem cells process.Real-time detection by cell tracker and cell differentiation procedure, can carry out real-time monitoring to the gel deposition of loading cell, thereby convenient research cell survives and differentiation and each parameter of deposition technique (as sedimentation velocity, piezoelectricity pulse width etc.) direct relation, shortened research cycle and cost greatly.
Compared with prior art, the present invention has following advantage:
(1) by stem cell and three-dimensional cell timbering material, the stratiform in syringe merges, and removal is injected the extraneous factor that cell causes after first making support, cell cultures and three-dimensional cell support are formed to two steps and separate, thereby a large amount of experiments is avoided in the substep realization; (2) by fluorescence and radiolabeled mode, mark is followed the tracks of culturing stem cells, thereby can determine and be mixed into injection again in deposition process, the content of stem cell in support and the distribution of stem cell, so just avoid utilizing microscopic examination test after poppet to form, there is real-time; (3) by utilizing the three-dimensional cell support to sneak into the mode of specialized cell proteolytic enzyme and detection test solution, can reach the purpose of real-time detection differentiation of stem cells situation, thereby, by obtaining about timbering material, the corresponding relation of structure and differentiation, improve in real time to follow-up print procedure.
Embodiment
Embodiment 1
(1) cell culture part:
(I) configuration neutral red staining liquid, get 0.1g guinea green B powder dissolution and stir in 1L physiological saline, the guinea green B solution that to be mixed with concentration be 0.01%.
(II) get the above-mentioned solution of 100ml and immerse in culture dish, after soaking bone stem cell 1h, by filter paper, filter out the bone stem cell.
(III) in the bone stem cell filtered out, by whizzer, separate, the cell of selective precipitation green, isolate without cytochrome.Wherein green cell is the bone stem cell that lives.Supplement again cell culture fluid and be configured to 100ml cell green solution.
(2) gel stent part:
The present invention adopts the basic material of seaweed gel material as support, mixes the alkaline phosphatase of phenolphthalein for detection of the scleroblast Early insulin secretion.
(I) get sodium alginate 4g and be dissolved in the 100ml deionized water, the gel stoste that to be mixed with weight percent concentration be 4%.
(II) get CaCl
2powder 2g is dissolved in 100ml distilled water, the gel body lotion that to be mixed with weight percent concentration be 2%.
(3) the bone stem cell of the green of (1) part screening is placed in to gel stoste, by the mode of aspirating, nucleus gel stoste is mixed, cultivate 4h.
(4) add the gel body lotion in the nutrient solution in (3), adition process is with stirring gently the stem cell that obtains the colloidal gel parcel.
(5) use filter paper filtering to go out the green cell of gel parcel, obtain bone stem cell and seaweed gel stratiform mixed structure, the stratiform hole of its seaweed gel, between 100-200um, carries out jet deposition in the reservoir of the 3 D-printing device of packing into.
(6) by CAD and the structure of CAM Software on Drawing design support and the movement locus of printing head, x by syringe and receiving plane, y, z tri-axles move the deposition injection and obtain definite three-dimensional rack structure arbitrarily, for example, needing the three-dimensional rack structure of printing in the present embodiment is the individual layer plane square structure, four bights that wherein Q, P, R, S are this square structure, receiving plane translational speed 10mm/s, air pressure 300kPa.Can select commercial extruding type type printer in the present embodiment.
(7) by the about 1min(of weak uviolizing, be medical science ultraviolet ray for sterilization), utilize alginates gel reversable light sensitive characteristic by the support solidifying and setting.
(8) by the supporting structure deposition process, add a small amount of distilled water flushing and filter, the distribution situation of Real Time Observation green is determined the distribution situation of bone stem cell, wherein, aobvious green part is the bone stem cell survived, and coloured moiety not shows that cell is dead or there is no a cell.Thereby determine the content information of different positions bone stem cell in the three-dimensional cell support by determining green the distribution.The individual layer plane square QPRS of take is example, and four angle Q, P, R, S place green is darker, and central point O(O is foursquare center, individual layer plane) locate the green fragmentary spot distribution that becomes.Illustrate, peripheral four jiaos of viable cell content are many and square center cell content is few.
(9) preparation phenolphthalein indicator (0.5% phenolphthalein ethanolic soln): get 0.5g phenolphthalein, use 95% dissolve with ethanol, and be diluted to 100mL, without adding water.
(10) support forms latter 24 hours, splash into phenolphthalein solution prepared by step (9) and observe the variation of observing color in support-individual layer plane square QPRS, containing red positional representation phenolphthalein reddens, produce alkaline phosphatase, thereby determine that key cytodifferentiation becomes the position of specialized cell, obtain the initial stage differentiation information of key cell.Observe, center O point redness is off and on, Q, P, R, the basic redfree in S place.Cell content few but successfully differentiation in center is described, the high but not well differentiation of under-nutrition of four horn cells content.
(11) support forms latter about 2 weeks, the content of collagen protein and distribution situation in Bracket for Inspection, thus determine the later stage differentiation information that obtains key cell, in Bracket for Inspection, the method for collagen protein is as follows:
(I) choose respectively foursquare four the angle Q of individual layer on the three-dimensional cell supporting structure, P, R, 5 of S and central point O are labeled as 1,2,3,4,5, and the sample 5g that gets five positions is dissolved in 50ml distilled water and is mixed with five parts of solution to be measured, is designated as a, b, c, d, o.
(II) use type i collagen ELISA detection kit, with after sample diluent 1:1 dilution, adding 50ml in reacting hole, then add testing sample 50ul in reacting hole.The biotin labeled antibody that adds immediately 50ul.Cover lamina membranacea, vibration mixes gently, 37 ℃ of incubations 1 hour.
(III) get rid of liquid in hole, washings is filled it up with in every hole, vibrates 30 seconds, gets rid of washings, with thieving paper, pats dry.Repeat this operation 3 times.If wash with washing the plate machine washing, the washing times increase once.
(IV) every hole adds affine chain enzyme-HRP of 80ml, and vibration mixes gently, 37 ℃ of incubations 30 minutes.
(V) get rid of liquid in hole, washings is filled it up with in every hole, vibrates 30 seconds, gets rid of washings, with thieving paper, pats dry.Repeat this operation 3 times.If wash with washing the plate machine washing, the washing times increase once.
(VI) every hole adds developer A, B(A, and B is that test kit carries developer) each 50ml, vibration mixes gently, 37 ℃ of incubations 10 minutes.Avoid illumination.
(VII) take out enzyme plate, add rapidly the 50ml stop buffer, add after stop buffer measurement result immediately.
(VIII) measure the OD value in each hole at 450nm wavelength place.Do not contain collagen protein if the OD value is extremely low or be zero.It is the not differentiation of cell of this position.
Claims (9)
1. the three-dimensional cell support Method of printing based on the cell state feedback, is characterized in that, comprising:
(1) culture dish that stem cell is placed in to vital dye is cultivated dyeing, until, after the stem cell variable color, coloured stem cell is separated with colourless stem cell, takes out coloured stem cell;
(2) the coloured stem cell after dyeing is placed in to gel stoste and cultivates, after having cultivated, add the gel body lotion, filter the stem cell that obtains the gel parcel;
(3) stem cell that step (2) is obtained to gel parcel is packed in the syringe of 3 D-printing device, carry out the printing of three-dimensional cell support, in print procedure, read in real time the distributed intelligence of coloured stem cell on the three-dimensional cell support according to the distribution situation of coloured stem cell, final curing obtains the three-dimensional cell support;
(4) the three-dimensional cell stentplacement that obtains, after 20-48 hour, is immersed in stem cell initial stage differentiation detection reagent, detects stem cell initial stage differentiation information on the three-dimensional cell support;
(5), by the three-dimensional cell stentplacement 10-15 days obtained, utilize stem cell later stage differentiation information on Protein Detection equipment Inspection three-dimensional cell support;
(6) finally make the following judgment:
Meet the requirements if prepare the three-dimensional cell support, complete print procedure;
If it is undesirable to prepare the three-dimensional cell support, on the three-dimensional cell support that on the three-dimensional cell support that the distributed intelligence of coloured stem cell on the three-dimensional cell support step (3) obtained, step (4) obtain, stem cell initial stage differentiation information and step (5) obtain, stem cell later stage differentiation information is as feedback information, 3 D-printing device print parameters in set-up procedure (3), repeating step (1)-(6).
2. the three-dimensional cell support Method of printing based on cell state feedback according to claim 1, it is characterized in that, in step (1), in described cultivation dyeing course, adopt the normal saline solution of guinea green B or toluylene red to be dyeed, the weight percent concentration of described guinea green B or toluylene red is 0.01-0.05%.
3. the three-dimensional cell support Method of printing based on cell state feedback according to claim 1, is characterized in that, in step (1), described coloured stem cell and colourless stem cell lock out operation adopted by centrifugation.
4. the three-dimensional cell support Method of printing based on cell state feedback according to claim 1, it is characterized in that, in step (2), described gel stoste is sodium alginate aqueous solution, and in described sodium alginate aqueous solution, the weight percent concentration of sodium alginate is 2-5%.
5. the three-dimensional cell support Method of printing based on the cell state feedback according to claim 4, is characterized in that the hydromining deionized water in described sodium alginate aqueous solution.
6. the three-dimensional cell support Method of printing based on the cell state feedback according to claim 1, is characterized in that, in step (2), described gel body lotion is CaCl
2the aqueous solution, described CaCl
2caCl in the aqueous solution
2mass percent concentration be 2-5%.
7. the three-dimensional cell support Method of printing based on cell state feedback according to claim 1, is characterized in that, in step (2), it is more than 4 hours that coloured stem cell is placed in the time that gel stoste cultivates.
8. the three-dimensional cell support Method of printing based on the cell state feedback according to claim 1, is characterized in that, in step (2), after adding the gel body lotion, keeps 0.5-3 minute.
9. the three-dimensional cell support Method of printing based on the cell state feedback according to claim 1, is characterized in that, in step (4), described stem cell initial stage differentiation detection reagent is 0.5% phenolphthalein ethanolic soln.
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