TW202246488A - Microcarriers with scaffold structure and continuous outer wall for culturing cells - Google Patents

Abstract

The invention relates to a microcarrier, comprising a continuous medium of a biocompatible polymer for culturing cells and having a three-dimensional scaffold architecture delineated peripherally by a continuous outer wall, in which spherical macropores are stacked to one another and interconnected by connecting pores. Exposure pores are formed on the continuous outer wall at positions where it is in contact with the macropores, through which the interior of the microcarrier may be in fluid communication with the ambient culture medium. The microcarrier herein is produced by cast-molding and, therefore, has a continuous outer wall which provides additional mechanical strength while maintaining the porosity. The microcarrier thus produced is configured in the form of a basic geometrical body. The invention further relates to a cast-molding process for producing the microcarrier.

Description

具有連續外壁及支架結構可用於培養細胞之微載體Microcarrier with continuous outer wall and scaffold structure for cell culture

本發明關於一種細胞培養裝置，特別是一種含有三維支架結構可用於培養細胞之微載體，其經由模鑄成型(mold casting)而具有連續外壁，從而保有高孔隙率並維持良好機械特性。本發明也關於這種三維支架微載體的模鑄成型方法。The present invention relates to a cell culture device, especially a microcarrier containing a three-dimensional scaffold structure for cell culture, which has a continuous outer wall through mold casting, thereby maintaining high porosity and maintaining good mechanical properties. The present invention also relates to the molding method of the three-dimensional scaffold microcarrier.

傳統上，活體外細胞培養是透過使細胞附著在組織培養塑膠器皿或胞外基質附著蛋白上，再給予適當的液體培養基促使其生長和增殖。然而，這種二維平面的培養方式與活體內的實際生理環境相去甚遠，無法模擬活體內細胞與胞外基質以及細胞彼此之間的交互作用，亦不利於重現諸如細胞移行、凋亡、轉錄調節和受體表達等複雜的細胞行為。二維培養方式更侷限了細胞的生長空間，不利於細胞的大量生產。三維細胞培養技術顯然是回應上述產業課題的較佳解決方案。Traditionally, in vitro cell culture is achieved by attaching cells to tissue culture plastic vessels or extracellular matrix attachment proteins, and then giving appropriate liquid medium to promote their growth and proliferation. However, this two-dimensional plane culture method is far from the actual physiological environment in vivo, and cannot simulate the interaction between cells and extracellular matrix and cells in vivo, and is not conducive to reproducing such things as cell migration, apoptosis, Complex cellular behaviors such as transcriptional regulation and receptor expression. The two-dimensional culture method further limits the growth space of cells, which is not conducive to the mass production of cells. Three-dimensional cell culture technology is obviously a better solution to respond to the above-mentioned industrial issues.

1980年代，美國勞倫斯伯克利國家實驗室的Mina Bissell博士在對乳腺癌的研究中開創了三維細胞培養的技術(請參見Petersen O.W., et al., PNAS, 89(19): 9064-9068 )，其涉及將細胞與具有三維結構的支架在活體外共同培養，使細胞在支架的三維立體空間結構中生長和移行。近年來，隨著細胞支架製作工序的成熟，三維細胞培養技術逐漸取代了傳統的二維平面培養技術而被廣泛地應用在組織工程等生物醫學領域中。在應用上，三維支架可於活體外或活體內供細胞生長、組織分化及重塑，最終產生具有實驗用途或進一步供用於移植之組織。在結構上，其為具有大量微小孔洞的堆疊結構體，以供細胞接種和附著，再藉此導引細胞朝依規劃的三維方向進行生長分化，產生擬似的再生組織或器官。 In the 1980s, Dr. Mina Bissell of the Lawrence Berkeley National Laboratory in the United States pioneered the technology of three-dimensional cell culture in the study of breast cancer (seeing Petersen OW, et al ., PNAS , 89(19): 9064-9068), its It involves co-cultivating cells with a three-dimensional scaffold in vitro, allowing cells to grow and migrate in the three-dimensional structure of the scaffold. In recent years, with the maturity of the cell scaffold manufacturing process, three-dimensional cell culture technology has gradually replaced the traditional two-dimensional planar culture technology and is widely used in biomedical fields such as tissue engineering. In terms of application, three-dimensional scaffolds can be used for cell growth, tissue differentiation and remodeling in vitro or in vivo, and finally produce tissues for experimental use or further for transplantation. Structurally, it is a stacked structure with a large number of tiny holes for cell seeding and attachment, and then guides the cells to grow and differentiate in the three-dimensional direction according to the plan, resulting in a simulated regenerative tissue or organ.

美國專利第8513014號敘述一種製造三維支架結構塊狀材的方法，其涉及使用一個氣泡產生器，將氣流導入基質流體中以產生尺寸均一的氣泡，使氣泡膠化後再減壓破泡，再經固化後可製成一塊具有三維支架結構的材料。美國專利早期公開案第2019/091690A1號和美國專利第10,828,635號分別敘述運用具有正交結構(T-junction)的微流道裝置來大量產生單分散性氣泡的裝置，以及將氣泡由液體中分離出來的分離裝置，促使氣泡排列成最密堆積狀態後再予以膠化、破泡、及固化後便能大量生產此類三維支架結構的塊狀材。美國專利申請案第17/184,276號敘述了一種運用高内相乳液模板工序來製作三維細胞支架的方法。其他類型的習用支架製備技術還包括鹽析法(salting-out process)、冷凍乾燥法(freeze drying process)以及固體自由成型法(solid freeform fabrication process)等。U.S. Patent No. 8513014 describes a method for manufacturing a three-dimensional scaffold structure block, which involves the use of a bubble generator to introduce airflow into the matrix fluid to generate bubbles of uniform size, decompress and break the bubbles after gelling, and then After curing, a piece of material with a three-dimensional scaffold structure can be made. U.S. Patent Early Publication No. 2019/091690A1 and U.S. Patent No. 10,828,635 respectively describe the use of a microchannel device with an orthogonal structure (T-junction) to generate a large number of monodisperse bubbles and separate the bubbles from the liquid The separation device that comes out can make the bubbles arrange into the most densely packed state and then gel, break the bubbles, and solidify to mass produce such three-dimensional scaffold structure blocks. US Patent Application No. 17/184,276 describes a method for fabricating three-dimensional cell scaffolds using a high internal phase emulsion templating process. Other types of commonly used stent fabrication techniques include salting-out process, freeze drying process, and solid freeform fabrication process.

雖然上述方法已經可以製作出三維支架的塊狀材，但於實際應用時需考慮細胞培養過程中新鮮培養液與代謝物的擴散速率能夠滿足所有附著於三維支架上的細胞健康地成長。因此塊狀材實質上必須細分成小顆粒來使用，  上述物質才能及時從微載體內部到外界之間來回進行擴散輸送。由過去經驗得知微載體的合適的尺寸一般可以在 500微米至3000微米範圍內選取。所選取的特定尺寸視所欲培養的細胞種類及培養條件而定。這個尺寸範圍內的微載體在持續攪動培養液時能始終保持懸浮流動狀態。Although the above method can already produce a three-dimensional scaffold block material, it is necessary to consider that the diffusion rate of fresh culture medium and metabolites in the cell culture process can satisfy the healthy growth of all cells attached to the three-dimensional scaffold. Therefore, the bulk material must be subdivided into small particles for use, so that the above-mentioned substances can be diffused and transported back and forth from the inside of the microcarrier to the outside in time. It is known from past experience that the suitable size of microcarriers can generally be selected within the range of 500 microns to 3000 microns. The particular size chosen will depend on the type of cells to be cultured and the culture conditions. Microcarriers in this size range can always maintain a suspended flow state when the culture medium is continuously agitated.

上述塊狀材細分成微載體的方法一般採用習知的機械高速切割。但是切割時微載體內部支架將會承受過高的瞬間機械應力，致使支架產生結構 缺陷，且外表形狀也因切割呈現破碎不規則，有大量的球狀巨孔裸露，不具有連續整齊的外壁。於細胞培養時的攪動狀態下，此種微載體因機械強度不足而容易崩解碎裂，其孔洞裸露的結構也使依附其上的細胞過度承受培養液流動時所造成的剪切力，從而導致細胞生產率低下。另外，高速切割所得的微載體尺寸分佈視切割方法而定。若要求尺寸均一性高則切割製程相對繁複；所費工時甚長。若求壓縮工時而簡化製程，則微載體尺寸分布甚大(數微米～千微米左右)。再經由篩選取其適合培養細胞者(最大與最小尺寸之比不超過1.5倍)，經驗顯示其產出效益(yield)甚低。 若採用另一習知技術：極短波長雷射切割。此方法仍需面對工序繁複、產品表面孔洞裸露、設備昂貴的問題。而雷射切痕耗損寬約0.1mm，設定最後微載體切割成1mm ³ 的尺寸，其產出效益粗估等於：(1/1.1) ³=75%，結果仍有25%的耗損。因此，相關技術領域對於具有高孔隙率但不致實質降低其機械強度的微載體及兼具低成本、低工時、低損耗的製造方法，仍然存在有高度的需求 。 The method for subdividing the bulk material into micro-carriers generally adopts known mechanical high-speed cutting. However, the internal support of the microcarrier will bear excessive instantaneous mechanical stress during cutting, resulting in structural defects of the support, and the appearance of the support is broken and irregular due to cutting, with a large number of spherical giant holes exposed, without a continuous and neat outer wall. Under the agitation state during cell culture, this kind of microcarrier is easy to disintegrate and fragment due to insufficient mechanical strength, and its exposed structure also makes the cells attached to it excessively bear the shear force caused by the flow of culture fluid, thus lead to low cell productivity. In addition, the size distribution of microcarriers obtained by high-speed cutting depends on the cutting method. If high dimensional uniformity is required, the cutting process is relatively complicated; it takes a long time. If the man-hour is reduced and the manufacturing process is simplified, the size distribution of the microcarriers is very large (about a few microns to a thousand microns). After screening to select those suitable for culturing cells (the ratio of the largest to the smallest size is not more than 1.5 times), experience shows that the yield is very low. If another known technology is used: very short wavelength laser cutting. This method still needs to face the problems of complicated procedures, exposed holes on the product surface, and expensive equipment. The loss width of the laser cutting marks is about 0.1mm. If the final microcarrier is cut to a size of 1mm ³ , the output benefit is roughly estimated to be (1/1.1) ³ =75%, and the result is still 25% loss. Therefore, there is still a high demand in the relevant technical field for microcarriers with high porosity without substantially reducing their mechanical strength and manufacturing methods with low cost, low man-hours, and low loss.

本發明揭露一種具有高孔隙率及良好機械特性，具有三維支架結構且可用於培養細胞之微載體，以及該微載體的新式製程。所述之微載體採用模鑄成型來製造，其涉及將一含有生物相容性高分子的泡沫體注入適當模具中，再予以破泡、固化成型，經過脫模後即獲得所述微載體。該泡沫體實質上由數目眾多的氣泡共存堆疊於含生物相容性高分子的溶液中所組成。在固化後氣泡所佔原有空間形成球狀巨孔，高分子溶液在固化後成為具有三維支架型態的連續媒質。相鄰巨孔接觸區因破泡過程產生連通孔。當泡沫體注入模具內而與模具表面以及外界的空氣接觸，由於高分子溶液的表面張力自然會形成一層溶液膜包圍此泡沫體。於固化後此溶液膜成為微載體外表的連續外壁。某些球狀巨孔會與連續外壁相鄰接觸，因破泡過程而在連續外壁上產生多個暴露孔。The invention discloses a microcarrier with high porosity and good mechanical properties, a three-dimensional scaffold structure and can be used for culturing cells, and a new manufacturing process of the microcarrier. The microcarrier is manufactured by mold casting, which involves injecting a foam containing biocompatible macromolecules into a suitable mold, then breaking the foam, solidifying and forming, and obtaining the microcarrier after demoulding. The foam is essentially composed of a large number of bubbles coexisting and stacking in a solution containing biocompatible polymers. After solidification, the original space occupied by the bubbles forms spherical macropores, and the polymer solution becomes a continuous medium with a three-dimensional scaffold after solidification. The contact area of adjacent macropores produces interconnected pores due to the bubble breaking process. When the foam is injected into the mold and contacts the surface of the mold and the outside air, due to the surface tension of the polymer solution, a solution film will naturally form to surround the foam. After curing the solution film becomes a continuous outer wall on the surface of the microcarriers. Some spherical macropores will be in adjacent contact with the continuous outer wall, and multiple exposed holes will be produced on the continuous outer wall due to the bubble breaking process.

因此，依據本發明的基本態樣，其在於提供一種微載體，其尺寸可以在500微米至3000微米之間作選擇，視所欲培養的細胞種類及培養條件而定。其整體外型呈現某種簡單的基本幾何體。其具有上述由連續媒質所構築之三維支架、多數個互相堆疊的球狀巨孔、連通孔、暴露孔以及連續外壁等形成微載體的結構特徵。每個球狀巨孔周圍可以和多個相鄰的球狀巨孔接觸並各別有連通孔相通。連續外壁與某些球狀巨孔接觸而形成暴露孔，經由該孔內部的支架結構和外界可以相通溶液。Therefore, according to the basic aspect of the present invention, it is to provide a microcarrier whose size can be selected between 500 microns and 3000 microns, depending on the type of cells to be cultured and the culture conditions. Its overall shape presents a certain simple basic geometry. It has the structural characteristics of the above-mentioned three-dimensional support constructed by the continuous medium, a plurality of spherical macropores stacked on each other, communicating holes, exposed holes and continuous outer walls, etc. to form a microcarrier. The periphery of each spherical macropore can be in contact with a plurality of adjacent spherical macropores and communicate with each other through communication holes. The continuous outer wall is in contact with some spherical macropores to form exposed pores, and the solution can communicate with the outside world through the scaffold structure inside the pores.

培養液與代謝物從微載體中心到邊界的擴散會經由球狀巨孔、連通孔、暴露孔等結構所形成的多條曲曲折折的路徑。這些結構都構成擴散瓶頸。為使擴散速率能滿足微載體內的細胞成長所需，擴散瓶頸不宜過多。依據發明人的實證經驗，如果以球狀巨孔的直徑作為1單位，則合宜的擴散距離約在3至5單位之間。因此，所述微載體的特徵尺寸與球狀巨孔的直徑的比值介於約6：1至約10：1。The diffusion of culture fluid and metabolites from the center to the boundary of the microcarrier will go through multiple tortuous paths formed by structures such as spherical giant pores, interconnected pores, and exposed pores. These structures all constitute diffusion bottlenecks. In order for the diffusion rate to be adequate for the growth of cells within the microcarriers, the diffusion bottleneck should not be excessive. According to the empirical experience of the inventors, if the diameter of the spherical macropore is taken as 1 unit, the appropriate diffusion distance is about 3 to 5 units. Thus, the ratio of the characteristic size of the microcarriers to the diameter of the spherical macropores is between about 6:1 and about 10:1.

依據本發明的另一態樣，其在於提供一種用於製造前述微載體的方法，其包含下列步驟： A、製備一個高分子泡沫體(polymeric foam)，其包含一連續相，以及一與該連續相不相混溶而且由許多個散佈於該連續相中的相互隔離單元構成的分散相，其中該連續相包含選自於由生物相容性高分子、其單體、其寡聚物和它們之組合所組成的群組的成份； B、將該高分子泡沫體填入一個多孔平板模具中，再將該高分子泡沫體固化而得到一連續媒質，其中該多孔平板模具界定出多個連接該多孔平板模具的兩個主要表面的微型通孔，且各個微型通孔被建構成具有基本幾何體的構形，並且具有一介於500微米至3000微米的特徵尺寸，以及其中該分散相中各個相互隔離單元具有的直徑與該特徵尺寸的比值介於約 1：6 至約 1：10；以及 C、使該連續媒質脫離該多孔平板模具，而得到具有三維細支架且有連續外壁用於培養細胞之微載體。 According to another aspect of the present invention, it is to provide a kind of method for making aforementioned microcarrier, and it comprises the following steps: A. Prepare a polymer foam (polymeric foam), which comprises a continuous phase, and a dispersed phase which is immiscible with the continuous phase and consists of many mutually isolated units dispersed in the continuous phase, wherein the The continuous phase comprises components selected from the group consisting of biocompatible macromolecules, monomers thereof, oligomers thereof, and combinations thereof; B. Fill the polymer foam into a porous flat mold, and then solidify the polymer foam to obtain a continuous medium, wherein the porous flat mold defines a plurality of holes connecting the two main surfaces of the porous flat mold Micro through holes, and each micro through hole is constructed to have a basic geometric configuration, and has a characteristic size between 500 micrometers and 3000 micrometers, and wherein each isolated unit in the dispersed phase has a diameter corresponding to the characteristic size a ratio of about 1:6 to about 1:10; and C. The continuous medium is separated from the porous plate mold to obtain a microcarrier with a three-dimensional fine support and a continuous outer wall for culturing cells.

在一較佳態樣中，前述微載體的暴露孔的孔徑實質小於與其相鄰的球狀巨孔的孔徑。In a preferred aspect, the diameter of the exposed pores of the aforementioned microcarriers is substantially smaller than the diameter of the adjacent spherical macropores.

在較佳的態樣中，該微載體中有至少50%的球狀巨孔是以最密堆積的形式排列。In a preferred aspect, at least 50% of the spherical macropores in the microcarrier are arranged in the form of closest packing.

在較佳的態樣中，所述微載體外型呈現之基本幾何體選自於由圓柱體、球體、圓錐體、正方體、長方體、稜柱體和稜錐體所組成的群組。在更佳的態樣中，所述基本幾何體選自於圓柱體。In a preferred aspect, the basic geometry of the appearance of the microcarrier is selected from the group consisting of cylinder, sphere, cone, cube, cuboid, prism and pyramid. In a more preferred aspect, the basic geometry is selected from cylinders.

在一較佳態樣中，所述生物相容性高分子選自於由蛋白質類、多醣類、人造高分子類，以及它們之組合所組成的群組。更佳為所述生物相容性高分子選自於由明膠、膠原蛋白、纖維蛋白(fibrins)、瓊脂糖、玻尿酸、甲殼素、海藻酸鹽、纖維素、吉蘭膠(gellan gum)所組成的群組。In a preferred aspect, the biocompatible polymer is selected from the group consisting of proteins, polysaccharides, artificial polymers, and combinations thereof. More preferably, the biocompatible polymer is selected from the group consisting of gelatin, collagen, fibrins, agarose, hyaluronic acid, chitin, alginate, cellulose, and gellan gum group.

本案所揭微載體及其製程的有益效果在於： 1、微載體內部結構的效益：內部球狀巨孔相互連通，構成了一個洞洞相連的連續網絡結構體，其具有極大的比表面積，適合大量細胞進入球狀巨孔內部並在巨孔孔壁上進行附著和生長。而新鮮培養液與代謝物可經由連通孔與暴露孔在微載體內部到外界之間來回進行快速擴散 ，進而促進細胞成長。 2、微載體外部連續壁的效益：於細胞培養時培養液持續攪動狀態下產生的亂流將對暴露在外的支架或細胞造成經常性的衝擊。具有實質平整的連續外壁可提供內部支架及細胞一層保護，減少亂流帶來的損壞。連續外壁對微載體內的支架具有結構上的連結及整合作用，可以均勻分散外界流體的剪切力，從而強化三維細胞支架的機械強度，避免微載體於細胞培養條件下崩解。 3、模鑄製程的簡化效益：如上述將泡沫體注入適當模具中，再予以破泡、固化、脫模後即可直接供作培養細胞的微載體。此製程可以減免以下製程及缺點： (1)、前期準備三維支架塊狀材的製作過程； (2)、高工時，高成本的機械或雷射切割塊狀材製程；(3)、塊狀材因切割造成高比例的損耗；以及(4)、切割造成微載體表面破損，導致微載體機械強度及細胞產量降低等副作用。因此可以大幅提昇產品效益。 4、模鑄新製程的系統性效益：所述模鑄新製程所用的模具形狀及尺寸可以最佳化設計，並精準複製大量模具於平面模版上。所製出的眾多微載體具有狹窄的尺寸分佈。有利於細胞培養的可控制性、可預測性、可分析性。 The beneficial effects of the microcarrier disclosed in this case and its manufacturing process are: 1. The benefits of the internal structure of the microcarrier: the internal spherical giant pores are connected to each other, forming a continuous network structure connected by holes. attach and grow on the wall. The fresh culture medium and metabolites can quickly diffuse back and forth between the inside of the microcarrier and the outside through the communicating pores and exposed pores, thereby promoting cell growth. 2. The benefits of the external continuous wall of the microcarrier: the turbulent flow generated by the continuous agitation of the culture medium during cell culture will cause frequent impacts on the exposed scaffolds or cells. Having a substantially flat continuous outer wall can provide a layer of protection for the inner scaffold and cells, reducing damage caused by turbulence. The continuous outer wall has a structural connection and integration effect on the scaffold in the microcarrier, and can evenly disperse the shear force of the external fluid, thereby strengthening the mechanical strength of the three-dimensional cell scaffold and preventing the microcarrier from disintegrating under cell culture conditions. 3. The benefits of simplification of the mold casting process: as mentioned above, inject the foam into a suitable mold, then break the foam, solidify, and demould, and then it can be directly used as a microcarrier for culturing cells. This process can reduce the following processes and disadvantages: (1), the pre-preparation process of the three-dimensional bracket block material; (2), high man-hours, high-cost mechanical or laser cutting block material process; (3), block The cutting causes a high proportion of loss of the shape material; and (4), the cutting causes damage to the surface of the microcarrier, resulting in side effects such as a decrease in the mechanical strength of the microcarrier and a reduction in cell yield. Therefore, the product efficiency can be greatly improved. 4. Systematic benefits of the new mold casting process: The shape and size of the molds used in the new mold casting process can be optimally designed, and a large number of molds can be accurately copied on the plane template. Numerous microcarriers produced have a narrow size distribution. Conducive to the controllability, predictability and analyzability of cell culture.

除非另行說明，否則本案專利說明書和各請求項中所使用的下列用語具有下文給予的定義。請注意，本案說明書和各請求項中所使用的單數形用語「一」意欲涵蓋在一個以及一個以上的所載事項，例如至少一個、至少二個或至少三個，而非意味著僅僅具有單一個所載事項。此外，本案各請求項中使用的「包含」、「具有」等開放式連接詞是表示請求項中所記載的元件之組合中，不排除請求項未載明的其他元件。亦應注意到用語「或」在意義上一般也包括「及/或」，除非內容另有清楚表明。本申請說明書和申請專利範圍中所使用的用語「 大約」或「實質上」，是用以修飾任何可些微變化的誤差，但這種些微變化並不會改變其本質。Unless otherwise stated, the following terms used in the patent specification and claims of this case have the definitions given below. Please note that the singular term "a" used in this case description and various claims is intended to cover one and more than one items, such as at least one, at least two or at least three, and does not mean only having a single A contained matter. In addition, open conjunctions such as "comprising" and "having" used in the claims of this case indicate the combination of elements described in the claims, and other elements not specified in the claims are not excluded. It should also be noted that the term "or" generally includes "and/or" unless the content clearly indicates otherwise. The term "approximately" or "substantially" used in the description of this application and the scope of the patent application is used to modify any error that may be slightly changed, but such a slight change will not change its essence.

圖1為本申請的一個實施例的示意圖，顯示微載體100主要包含一連續媒質120。本說明書中所稱「連續媒質」是指由生物相容性高分子所構成的單件式（monolithic）媒介物質，適合細胞附著於上並且進行生長、增殖和移行。此處所稱「生物相容性」是用於描述不會對於細胞、組織和器官等生物系統引起不良副用的材料。適用於製作微載體的生物相容性高分子已為相關業界所熟悉，其包括但不限於：蛋白質類，例如明膠、膠原蛋白、纖維蛋白(fibrins)等；多醣類，例如瓊脂糖、玻尿酸、甲殼素、海藻酸鹽、纖維素、吉蘭膠(gellan gum)等；人造高分子類，例如聚酯醯胺類、聚己內酯多元醇(PCL)、聚乳酸(PLA)聚甘醇酸(PGA)、聚乳酸-羥基乙酸共聚物(PLGA)等具有生物可降解性的高分子，以及例如聚雙甲基矽氧烷(PDMS)、熱塑性聚胺酯、聚對苯二甲酸二乙酯(PET)、聚四氟乙烯(PTFE)、聚偏二氟乙烯(PVDF)、聚苯乙烯等不具生物可降解性的高分子；以及它們之組合。在圖2A至2B、圖3A至3B、圖4A至4B和圖6所顯示的較佳具體例中，微載體100皆是由膠原蛋白製成。FIG. 1 is a schematic diagram of an embodiment of the present application, showing that a microcarrier 100 mainly includes a continuous medium 120 . The term "continuous medium" in this specification refers to a monolithic medium composed of biocompatible polymers, which is suitable for cells to attach to and grow, proliferate and migrate. The term "biocompatibility" here is used to describe materials that do not cause adverse side effects to biological systems such as cells, tissues and organs. Biocompatible polymers suitable for making microcarriers are well known in the relevant industries, including but not limited to: proteins, such as gelatin, collagen, fibrins, etc.; polysaccharides, such as agarose, hyaluronic acid , chitin, alginate, cellulose, gellan gum, etc.; artificial polymers, such as polyester amides, polycaprolactone polyol (PCL), polylactic acid (PLA) polyglycol Acid (PGA), polylactic-co-glycolic acid (PLGA) and other biodegradable polymers, as well as polydimethylsiloxane (PDMS), thermoplastic polyurethane, polyethylene terephthalate ( PET), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polystyrene and other non-biodegradable polymers; and their combinations. In the preferred embodiments shown in FIGS. 2A to 2B , FIGS. 3A to 3B , FIGS. 4A to 4B and FIG. 6 , the microcarriers 100 are all made of collagen.

如圖1、2A至2B和3A至3B所示，微載體100具有一實質上呈現基本幾何體之構形。此處所稱「基本幾何體」意指由簡單之曲面及/或平面所組成的立體結構，其包括：圓柱體、球體、圓錐體等曲面幾何體，以及正方體、長方體、稜柱體、稜錐體等平面幾何體。微載體100可以透過模鑄成型而被建構成具有任何基本幾何體之構形，只要便於模鑄和脫模作業即可。在一較佳具體例中，微載體100被建構成圓柱體之構形。如後文所述，微載體100是透過具有固定尺寸的模具模鑄成型，因而具有狹窄的尺寸分佈。依據本發明，微載體100具有一介於500微米至3000微米的特徵尺寸，視所欲培養的細胞種類及培養條件而定。所處所稱「特徵尺寸(characteristic dimension)」意指用於描述微載體100之外觀的最大尺度，例如微載體100的長、寬、高、直徑等。舉例來說，圓柱形微載體的特徵尺寸可以指涉它的高或直徑。在本申請中，「特徵尺寸」也用於描述模具的微型通孔的最大尺度，例如微型通孔的孔徑和高度。因此，本發明透過設定模具的尺寸，使得微載體100被製造成具有一介於500微米至3000微米的特徵尺寸，以利懸浮於液態細胞培養液中。在一具體例中，微載體100具有一介於500微米至880微米的特徵尺寸，使得微載體100的特徵尺寸小於工業用和實驗室用定量吸管的管徑，因而適合於透過習用吸管取得定量樣品，有利於快速檢測細胞生長及調控生長條件。微載體100的特徵尺寸可以藉由在電子顯微鏡下測量，如圖3A所示。所述特徵尺寸也可以經由過篩來測量，亦即，微載體100能夠通過依據泰勒標準篩制(Tyler standard screen scale)為6目篩網(篩孔尺寸3350微米)，甚至通過18目篩網(篩孔尺寸880微米)，但不能通過32目篩網(篩孔尺寸500微米)。As shown in Figures 1, 2A-2B and 3A-3B, the microcarrier 100 has a configuration that substantially exhibits a basic geometry. The term "basic geometry" here refers to a three-dimensional structure composed of simple curved surfaces and/or planes, including: cylinders, spheres, cones and other curved surfaces, and cubes, cuboids, prisms, pyramids and other planes geometry. The microcarriers 100 can be constructed by molding to have any basic geometric configuration as long as the molding and demolding operations are convenient. In a preferred embodiment, the microcarrier 100 is constructed in the shape of a cylinder. As will be described later, the microcarriers 100 are molded through a mold with fixed dimensions and thus have a narrow size distribution. According to the present invention, the microcarrier 100 has a characteristic size ranging from 500 microns to 3000 microns, depending on the type of cells to be cultured and the culture conditions. The term "characteristic dimension" refers to the largest dimension used to describe the appearance of the microcarrier 100 , such as the length, width, height, and diameter of the microcarrier 100 . For example, a characteristic dimension of a cylindrical microcarrier can refer to its height or diameter. In this application, "feature size" is also used to describe the maximum dimension of the micro-vias of the mold, such as the diameter and height of the micro-vias. Therefore, in the present invention, by setting the size of the mold, the microcarrier 100 is manufactured to have a characteristic size ranging from 500 microns to 3000 microns, so as to be suspended in the liquid cell culture solution. In a specific example, the microcarrier 100 has a characteristic size between 500 microns and 880 microns, so that the characteristic size of the microcarrier 100 is smaller than the diameter of quantitative pipettes used in industry and laboratories, thus suitable for obtaining quantitative samples through conventional pipettes , which is conducive to rapid detection of cell growth and regulation of growth conditions. The characteristic size of the microcarrier 100 can be measured under an electron microscope, as shown in FIG. 3A . The characteristic size can also be measured by sieving, i.e. the microcarriers 100 can pass through a 6 mesh screen (mesh size 3350 microns) according to the Tyler standard screen scale, even through an 18 mesh screen (mesh size 880 microns), but cannot pass through a 32-mesh sieve (mesh size 500 microns).

如圖1和圖4A至4B所示，微載體100形成有數個球狀巨孔121。如後文所述，球狀巨孔121的尺寸可以透過調整所述高分子泡沫體的製造工序的參數條件，經由調控分散相中相互隔離單元(例如氣泡或微液滴)的尺寸，而使得球狀巨孔121的直徑與微載體100的特徵尺寸的比值介於約 1：6 至約 1：10，以利於微載體內部與外界的物質透過擴散作用而傳送。例如，特徵尺寸約1000微米的微載體內所含球狀巨孔的直徑約在100微米至170微米，而特徵尺寸約3000微米的微載體內所含球狀巨孔的直徑約在300微米至500微米。在一具體例中，球狀巨孔121被製作成具有介於5微米至500微米的直徑，較佳為具有介於50微米至200微米的直徑。球狀巨孔121相互鄰接。此處所稱「相互鄰接」意指微載體中的一個球狀巨孔，通常與至少另一個球狀巨孔透過至少一個連通孔相通。連通孔122的尺寸可以透過在製造上述泡沫體的工序中調節分散相中相鄰相互隔離單元(例如氣泡或微液滴)之間的接觸面積來進行控制。在一些具體例中，所有的球狀巨孔121皆具有實質上均一的直徑，而在其他具體例中，球狀巨孔121的尺寸分佈較為寬廣，取決於上述氣泡或微液滴是否具有單分散性而定。在一具體例中，微載體100中至少有一部分的球狀巨孔121呈有序排列，例如以最密堆積的形式排列。較佳為微載體100中有至少50%的球狀巨孔121，更佳為有至少60%的球狀巨孔121，最佳為有至少70%的球狀巨孔121，例如有至少80%的球狀巨孔121，是以最密堆積的形式排列。As shown in FIG. 1 and FIGS. 4A to 4B , the microcarrier 100 is formed with several spherical macropores 121 . As described later, the size of the spherical macropores 121 can be adjusted by adjusting the parameters of the polymer foam manufacturing process, and by controlling the size of the mutually isolated units (such as air bubbles or micro-droplets) in the dispersed phase, so that The ratio of the diameter of the spherical macropore 121 to the characteristic size of the microcarrier 100 is about 1:6 to about 1:10, so as to facilitate the transfer of substances inside and outside the microcarrier through diffusion. For example, the diameter of spherical megapores contained in a microcarrier with a characteristic size of about 1000 microns is about 100 microns to 170 microns, while the diameter of spherical macropores contained in a microcarrier with a characteristic size of about 3000 microns is about 300 microns to 300 microns. 500 microns. In a specific example, the spherical macropore 121 is made to have a diameter ranging from 5 microns to 500 microns, preferably having a diameter ranging from 50 microns to 200 microns. The spherical macropores 121 are adjacent to each other. The term "mutually adjacent" here means that one spherical macropore in the microcarrier usually communicates with at least one other spherical macropore through at least one connecting hole. The size of the communication holes 122 can be controlled by adjusting the contact area between adjacent isolated units (such as air bubbles or micro-droplets) in the dispersed phase during the manufacturing process of the above-mentioned foam. In some embodiments, all the spherical macropores 121 have a substantially uniform diameter, while in other embodiments, the size distribution of the spherical macropores 121 is relatively broad, depending on whether the above-mentioned bubbles or micro-droplets have a single diameter. Depends on dispersion. In a specific example, at least a part of the spherical macropores 121 in the microcarrier 100 are arranged in an orderly manner, for example arranged in a form of closest packing. Preferably there are at least 50% spherical macropores 121 in the microcarrier 100, more preferably at least 60% spherical macropores 121, most preferably at least 70% spherical macropores 121, such as at least 80% % of the spherical megapores 121 are arranged in the form of the closest packing.

微載體100具有一連續外壁123。以微載體100被建構成圓柱體構形為例，其具有由一頂部平面、一底部平面和一側曲面所組成的連續外壁123，如圖1、2A至2B和3A至3B所示。此處所稱「連續外壁」意指外壁中的所有位點都直接地相接不中斷。連續外壁123使得連續媒質120的結構完整，並且可以強化微載體100的機械強度，避免於細胞培養條件下崩解。連續外壁123也使得球狀巨孔121不致完全裸露，可以保護附著於三維細胞支架100內部的細胞，使它們免於承受細胞培養條件下因培養液流動時所造成的剪切力，從而提供細胞生長環境的穩定性。Microcarrier 100 has a continuous outer wall 123 . Taking the microcarrier 100 constructed as a cylindrical shape as an example, it has a continuous outer wall 123 composed of a top plane, a bottom plane and a curved side, as shown in FIGS. 1 , 2A-2B and 3A-3B. The term "continuous outer wall" herein means that all points in the outer wall are directly connected without interruption. The continuous outer wall 123 makes the structure of the continuous medium 120 complete, and can enhance the mechanical strength of the microcarrier 100 to avoid disintegration under cell culture conditions. The continuous outer wall 123 also prevents the spherical megapore 121 from being completely exposed, which can protect the cells attached to the inside of the three-dimensional cell scaffold 100, and prevent them from bearing the shear force caused by the flow of the culture solution under cell culture conditions, thereby providing cells Stability of the growing environment.

如圖1、2A至2B和3A至3B所示，連續外壁123形成有複數個與外界連通的暴露孔124，將球狀巨孔121部分地暴露出來，亦即，暴露孔124的孔徑實質小於與其相鄰的球狀巨孔121的孔徑，使得球狀巨孔121不致完全裸露。接受培養的細胞可以經由暴露孔124而進入微載體100內部。As shown in Figures 1, 2A to 2B and 3A to 3B, the continuous outer wall 123 is formed with a plurality of exposure holes 124 communicating with the outside world, and the spherical megapore 121 is partially exposed, that is, the diameter of the exposure holes 124 is substantially smaller than The diameter of the adjacent spherical macropore 121 prevents the spherical macropore 121 from being completely exposed. Cells to be cultured can enter the interior of the microcarrier 100 through the exposure hole 124 .

圖5是本發明用於製造微載體的方法的流程圖，其包含步驟A：製備一個高分子泡沫體；步驟B：將該高分子泡沫體加以模鑄成型為連續媒質；以及步驟C：使連續媒質脫模。Fig. 5 is the flowchart of the method for making microcarrier of the present invention, and it comprises step A: prepare a polymer foam; Step B: this polymer foam is molded into continuous medium; And step C: make Continuous media release.

步驟A包含製備一個高分子泡沫體，其包含一連續相，以及一與該連續相不相混溶且由許多散佈於該連續相中的相互隔離單元構成的分散相。依據本發明，連續相是發生固化反應而生成連續媒質的相，其包含選自於由前述生物相容性高分子、其單體、其寡聚物和它們之組合所組成的群組的成份。連續相還可以包含固化反應所需要的其他成份，例如交聯劑、聚合反應起始劑、乳化安定劑、界面活性劑、鹽類、溶劑等。連續相在常溫下通常呈黏稠流體。此處所稱「固化」意指對於呈流體狀態的連續相施以物理性或化學性架橋手段，從而將其轉化成為一具有穩定固體構形的連續媒質之過程。在某些具體例，分散相是氣體，而連續相是油性或水性的溶液或懸浮液。在另外的具體例中，分散相是水性溶液，而所述高分子泡沫體呈現油包水乳液(water-in-oil emulsion)的形式。Step A involves preparing a polymer foam comprising a continuous phase and a dispersed phase immiscible with the continuous phase and composed of a plurality of mutually isolated units dispersed in the continuous phase. According to the present invention, the continuous phase is a phase in which a solidification reaction occurs to form a continuous medium, which includes components selected from the group consisting of the aforementioned biocompatible polymers, their monomers, their oligomers, and combinations thereof . The continuous phase may also contain other components needed for the curing reaction, such as cross-linking agent, polymerization initiator, emulsion stabilizer, surfactant, salt, solvent and so on. The continuous phase is usually a viscous fluid at room temperature. The term "solidification" here refers to the process of applying physical or chemical bridging means to the continuous phase in a fluid state, thereby transforming it into a continuous medium with a stable solid configuration. In some embodiments, the dispersed phase is a gas and the continuous phase is an oily or aqueous solution or suspension. In another embodiment, the dispersed phase is an aqueous solution, and the polymer foam is in the form of a water-in-oil emulsion.

用於製作上述泡沫體的手段可見於美國專利第8513014號和美國專利早期公開案第2019/091690A1號，其涉及運用多相流法(multiphase flow)，將氣流或液流經由微流道裝置導入連續相中，透過對微流道裝置的特殊設計以及對流體流速的控制，產生散佈於連續相中的氣泡或微液滴。應用上述專利文件所揭露的手段，可以藉由改變微流道的尺寸和幾何形狀、流體的性質(例如黏度、表面張力)和流速，而大量製造出單分散性氣泡或微液滴，還可以進一步使它們排列成最密堆積狀態，用以製造具有均一尺寸的球狀巨孔的微載體。上述專利和專利申請案均通過引用而完整地併入本說明書。The means for making the above-mentioned foams can be found in U.S. Patent No. 8513014 and U.S. Patent Early Publication No. 2019/091690A1, which involve the use of multiphase flow (multiphase flow) to introduce gas or liquid flow through a microfluidic device. In the continuous phase, through the special design of the micro-channel device and the control of the fluid flow rate, bubbles or micro-droplets dispersed in the continuous phase are generated. By applying the means disclosed in the above patent documents, a large number of monodisperse bubbles or micro-droplets can be produced by changing the size and geometry of the micro-channel, the properties of the fluid (such as viscosity, surface tension) and flow velocity, and can also They are further arranged into the closest packing state to manufacture microcarriers with spherical macropores of uniform size. The foregoing patents and patent applications are hereby incorporated by reference in their entirety.

另一種製作泡沫體的手段涉及將連續相組成物與不相混溶的分散相組成物，以高轉速的均質機進行劇烈攪拌，使分散相均勻分散於連續相中，從而獲得一個油包水乳液。可以選擇性地進一步使所述油包水乳液接受外力沈降，以提高乳液中分散相相對於連續相的體積比，而得到一高內相乳液，從而提昇所製成的微載體的孔隙度並且增加連通孔的尺寸。如相關技術領域中具有通常知識者所熟知，可以透過改變乳液中分散相相對於連續相的體積比以及擾動的速率和溫度等參數，來調整分散相中的微液滴的尺寸和均一度。Another method of making foam involves vigorously stirring the continuous phase composition and the immiscible dispersed phase composition with a high-speed homogenizer to uniformly disperse the dispersed phase in the continuous phase, thereby obtaining a water-in-oil lotion. The water-in-oil emulsion can optionally be further subjected to external force settling, to improve the volume ratio of the dispersed phase in the emulsion relative to the continuous phase, and obtain a high internal phase emulsion, thereby promoting the porosity of the microcarrier made and Increase the size of the connecting hole. As is well known to those skilled in the relevant art, the size and uniformity of the micro-droplets in the dispersed phase can be adjusted by changing the volume ratio of the dispersed phase to the continuous phase in the emulsion as well as the rate and temperature of agitation.

可供用於製作泡沫體的其他方法，亦適用於本發明。Other methods available for making foams are also suitable for use in the present invention.

步驟B中，先準備一個多孔平板模具，其界定出多個連接該多孔平板模具的兩個主要表面的微型通孔，較佳為這些微型通孔排列成陣列。這些通孔具有一實質上呈現基本幾何體之構形，亦即，它們具有一選自於由圓柱體、球體、圓錐體、正方體、長方體、稜柱體和稜錐體所組成之群組的構形。在圖6所顯示的較佳具體例中，這些通孔被建構成具有圓柱體之構形。各個微型通孔具有一介於500微米至3000微米的特徵尺寸，亦即，微型通孔的高度(相當於多孔平板模具的厚度)位於500微米至3000微米的範圍內，以及/或是微型通孔的孔徑位於500微米至3000微米的範圍內。在一具體例中，各個微型通孔被建構成具有一介於500微米至880微米的特徵尺寸。所述多孔平板模具可以由任何不會與高分子泡沫體產生物理和化學反應的惰性材料所製成，例如碳纖維、陶瓷、玻璃、石英、或是聚氯乙烯(PVC)、聚甲醛(POM)、聚碳酸酯(PC)、聚苯醚(PPO)、PA6/66尼龍塑膠、聚碳酸酯/丙烯腈-丁二烯-苯乙烯共聚物(PC/ABS)複合塑膠、聚對苯二甲酸酯(PET)、聚乙烯亞胺(PEI)、聚甲基丙烯酸甲酯(PMMA)、聚苯硫醚(PPS)、聚乙烯(PE)、聚丙烯(PP)、聚苯乙烯(PS)、乙烯/醋酸乙烯酯共聚物(EVA)等塑膠材料，抑或是不銹鋼、鈦、鋁、鋁鎂合金等金屬材料所製成。In step B, a porous plate mold is firstly prepared, which defines a plurality of micro through-holes connecting the two main surfaces of the porous plate mold, preferably these micro-through holes are arranged in an array. The through-holes have a substantially geometric configuration, that is, they have a configuration selected from the group consisting of cylinders, spheres, cones, cubes, cuboids, prisms and pyramids . In the preferred embodiment shown in FIG. 6, the through-holes are configured to have a cylindrical configuration. Each micro-via has a characteristic size between 500 microns and 3000 microns, that is, the height of the micro-vias (equivalent to the thickness of the porous plate mold) is in the range of 500 microns to 3000 microns, and/or the micro-vias The pore size lies in the range of 500 microns to 3000 microns. In one embodiment, each microvia is configured to have a feature size between 500 microns and 880 microns. The porous plate mold can be made of any inert material that does not react physically and chemically with the polymer foam, such as carbon fiber, ceramics, glass, quartz, or polyvinyl chloride (PVC), polyoxymethylene (POM) , polycarbonate (PC), polyphenylene oxide (PPO), PA6/66 nylon plastic, polycarbonate/acrylonitrile-butadiene-styrene copolymer (PC/ABS) composite plastic, polyethylene terephthalate Polyester (PET), Polyethyleneimine (PEI), Polymethylmethacrylate (PMMA), Polyphenylene Sulfide (PPS), Polyethylene (PE), Polypropylene (PP), Polystyrene (PS), Plastic materials such as ethylene/vinyl acetate copolymer (EVA), or metal materials such as stainless steel, titanium, aluminum, and aluminum-magnesium alloys.

依據本發明，上述泡沫體的分散相中各個相互隔離單元的直徑被調整成與微型通孔的特徵尺寸的比值介於約 1：6 至約 1：10。在一具體例中，各個相互隔離單元具有介於5微米至500微米的直徑，較佳為具有介於150微米至200微米的直徑。According to the present invention, the diameter of each isolated unit in the dispersed phase of the foam is adjusted to have a ratio of about 1:6 to about 1:10 to the characteristic size of the micro-through hole. In one embodiment, each isolated unit has a diameter between 5 microns and 500 microns, preferably between 150 microns and 200 microns.

多孔平板模具的製作工序為相關技術領域中具有通常知識者所熟悉，而且可以依據選用材質的不同而進行調整。舉例而言，當模具是由塑膠材料製成時，適用的製作工序包括但不限於射出成型、壓模成型、熱成型等塑膠加工製程，後續可搭配習用衝壓打孔或鑽孔製程製作微型通孔。當模具是由金屬材料製成時，可以藉由衝壓、輾軋、壓模成型、鍛造等慣用金屬加工製程來製作，隨後可任擇地搭配習用衝壓打孔或鑽孔製程製作微型通孔。The manufacturing process of the porous flat plate mold is familiar to those with ordinary knowledge in the relevant technical field, and can be adjusted according to the different materials selected. For example, when the mold is made of plastic materials, applicable manufacturing processes include but are not limited to plastic processing processes such as injection molding, compression molding, and thermoforming. hole. When the mold is made of metal material, it can be produced by conventional metal processing processes such as stamping, rolling, compression molding, forging, etc., and then optionally combined with conventional punching or drilling processes to make micro-through holes.

將步驟A所製得的高分子泡沫體倒在多孔平板模具上，再使用塑膠刮刀以適當速度刮過模具表面，使泡沫體受擠壓而進入且填滿微型通孔。模具下方若有外溢之泡沫體，可用刮刀進一步刮除。由於泡沫體密度極低，其對於微型通孔內壁的附著力足以將本身維持在通孔內而不致在固化前流失。隨後將組成物加以固化。此處所稱「固化」意指對於呈流體狀態的連續相施以物理性或化學性架橋手段，從而將其轉化成為一具有穩定固體構形的連續媒質之過程。固化的反應條件視高分子的類型不同而有差異，但皆為相關業界所熟悉。舉例來說，在使用膠原蛋白或明膠作為生物相容性高分子的具體例中，可以使填入模具中的組成物於低溫脫水，從而膠化定形。在使用海藻酸鹽作為生物相容性高分子的具體例中，可以加入含有鈣離子或鎂離子等二價金屬離子的溶液，使海藻酸鹽分子之間發生交聯反應，從而膠化定形。在使用聚苯乙烯作為生物相容性高分子的具體例中，可以促使連續相中的苯乙烯單體發生自由基聚合反應而固化。可以繼續將固化完成的連續媒質加以冷凍乾燥，較佳為在真空下進行冷凍乾燥，有助於使分散相中的氣泡或微液滴破裂而生成連通孔和暴露孔。Pour the polymer foam prepared in step A onto a porous flat mold, and then use a plastic scraper to scrape across the surface of the mold at an appropriate speed, so that the foam is squeezed to enter and fill the micro through holes. If there is overflowing foam under the mold, it can be further scraped off with a scraper. Due to the extremely low density of the foam, its adhesion to the inner walls of the micro-holes is sufficient to maintain itself in the holes without being lost before curing. The composition is then cured. The term "solidification" here refers to the process of applying physical or chemical bridging means to the continuous phase in a fluid state, thereby transforming it into a continuous medium with a stable solid configuration. The curing reaction conditions vary depending on the type of polymer, but they are all familiar to the relevant industries. For example, in the case where collagen or gelatin is used as the biocompatible polymer, the composition filled into the mold can be dehydrated at low temperature to gel and set. In a specific example of using alginate as a biocompatible polymer, a solution containing divalent metal ions such as calcium ions or magnesium ions can be added to cause a cross-linking reaction between alginate molecules, thereby gelling and setting. In a specific example where polystyrene is used as the biocompatible polymer, the styrene monomer in the continuous phase can be cured by free radical polymerization. The solidified continuous medium can be further freeze-dried, preferably under vacuum, which helps to break the bubbles or micro-droplets in the dispersed phase to form interconnected pores and exposed pores.

圖6顯示在模具200中經過冷凍乾燥的膠原蛋白泡沫體，其中去除分散相125所遺留下來空間將會成為連續媒質中的球狀巨孔。值得注意的是，由圖6可以看出，分散相125與模具200之間因連續相液體126的內聚力仍會留存有一層稀薄的連續相液體126，而此一薄層於固化後即形成連續外壁123。如此形成的連續外壁123與模具200在形狀上實質互補，使得連續媒質120的結構完整，強化其機械強度，避免於細胞培養條件下崩解。於冷凍乾燥期間，球狀巨孔與連續外壁123相接處的薄弱部分，將會因連續媒質120的內外壓力不均衡而破裂，從而形成暴露孔。這些暴露孔的孔徑實質小於與其相鄰的球狀巨孔的孔徑，不致使球狀巨孔完全裸露。固化反應完成後可以得到類似海綿狀或蜂巢狀的連續媒質，其內部具有大量適於細胞附著和生長的球形巨孔。FIG. 6 shows a freeze-dried collagen foam in a mold 200, in which the space left by removal of the dispersed phase 125 will become spherical macropores in the continuous medium. It is worth noting that, as can be seen from FIG. 6, a thin layer of continuous phase liquid 126 remains between the dispersed phase 125 and the mold 200 due to the cohesive force of the continuous phase liquid 126, and this thin layer forms a continuous phase after curing. The outer wall 123 . The thus formed continuous outer wall 123 is substantially complementary in shape to the mold 200, making the structure of the continuous medium 120 complete, strengthening its mechanical strength, and avoiding disintegration under cell culture conditions. During the freeze-drying process, the weak part where the spherical megapore meets the continuous outer wall 123 will be ruptured due to the unbalanced internal and external pressure of the continuous medium 120 , thereby forming an exposed hole. The pore diameters of these exposed pores are substantially smaller than those of the adjacent spherical macropores, so that the spherical macropores are not completely exposed. After the curing reaction is completed, a sponge-like or honeycomb-like continuous medium can be obtained, with a large number of spherical macropores suitable for cell attachment and growth inside.

步驟C可以使用任何能夠讓連續媒質120在其結構不受到實質損傷的前提下脫離模具的脫模工序，例如可以使用高壓空氣將連續媒質120吹出。在使用膠原蛋白和明膠作為生物相容性高分子的具體例中，可以將脫模後的連續媒質120在高於37℃的溫度下加熱進行乾燥和熱架橋。例如可以將由膠原蛋白所製成的連續媒質120置入烤箱( DENG YNG DO60型)，於50℃下真空乾燥1小時，隨後於150℃下加熱12至48小時，得到大量、尺寸分佈狹窄且具有連續外壁的微載體100。Step C can use any demoulding process that can make the continuous medium 120 out of the mold without substantial damage to its structure, for example, high-pressure air can be used to blow out the continuous medium 120 . In the specific example of using collagen and gelatin as the biocompatible polymer, the continuous medium 120 after demolding can be heated at a temperature higher than 37° C. for drying and thermal bridging. For example, the continuous medium 120 made of collagen can be placed in an oven (DENG YNG DO60 type), vacuum-dried at 50°C for 1 hour, and then heated at 150°C for 12 to 48 hours to obtain a large amount of collagen with a narrow size distribution and Microcarrier 100 with continuous outer wall.

圖7顯示經過傳統切削工序所製成的微載體，其外形破碎且尺寸不一，而圖2A至2B和圖3A至3B顯示本發明的微載體呈現完整的基本幾何體構形，而且展現出狹窄的尺寸分佈。本發明的微載體具有優異的機械強度。經過實際細胞培養條下進行驗證，本發明的微載體在攪拌式生物反應器系統中經歷超過14天的攪拌，仍不會發生分解碎裂的現象。Fig. 7 shows the microcarriers made by the traditional cutting process, its shape is broken and different in size, while Fig. 2A to 2B and Fig. 3A to 3B show that the microcarriers of the present invention present a complete basic geometric configuration, and exhibit narrow size distribution. The microcarriers of the present invention have excellent mechanical strength. After verification under actual cell culture conditions, the microcarrier of the present invention will not decompose and fragment after being stirred in a stirred bioreactor system for more than 14 days.

本發明的微載體在組織工程、腫瘤學、再生醫學、藥物篩選測試和幹細胞生物學均有廣泛的應用。本發明的微載體具有高機械強度、高比表面積和高孔洞連通性的優勢，適合於活體外與各種細胞共同培養以大量生產細胞，抑或於活體內供植入的細胞生長以重塑受損的組織。在使用蛋白質類或多醣類作為支架材料的具體例中，也可以使用例如胰蛋白酶等適當酵素，溶解微載體以回收細胞。The microcarrier of the present invention has wide applications in tissue engineering, oncology, regenerative medicine, drug screening test and stem cell biology. The microcarrier of the present invention has the advantages of high mechanical strength, high specific surface area and high pore connectivity, and is suitable for co-cultivation with various cells in vitro to produce cells in large quantities, or for implanted cell growth in vivo to remodel damaged cells organization. In specific examples where proteins or polysaccharides are used as scaffold materials, appropriate enzymes such as trypsin can also be used to dissolve microcarriers to recover cells.

以上諸實施例僅供說明本發明之用，而並非對本發明的限制，相關領域的技術人員，在不脫離本發明的技術範圍做出的各種修改或變化也應屬於本發明的保護範疇。The above embodiments are only for illustrating the present invention, rather than limiting the present invention. Various modifications or changes made by those skilled in the relevant fields without departing from the technical scope of the present invention should also belong to the protection category of the present invention.

100:微載體 120:連續媒質 121:球狀巨孔 122:連通孔 123:連續外壁 124:暴露孔 125:分散相 126:連續相液體 200:模具 100: Microcarriers 120: Continuous media 121: spherical giant hole 122: Connecting hole 123: continuous outer wall 124: Exposure hole 125: dispersed phase 126: continuous phase liquid 200: Mold

圖1為依據本發明一具體例的微載體的示意圖，其顯示圓柱形微載體的剖面二維結構，其中連續媒質和連續外壁在高分子泡沫體固化成形後是呈現連續一體的結構，為了說明方便，刻意區分為兩個元件；Fig. 1 is the schematic diagram according to the microcarrier of a specific example of the present invention, and it shows the cross-sectional two-dimensional structure of cylindrical microcarrier, and wherein continuous medium and continuous outer wall are to present the structure of a continuous body after polymer foam is solidified and formed, in order to illustrate Convenience, deliberately divided into two components;

圖2A和2B分別為依據本發明一具體例所製成的微載體的電子顯微鏡照片和示意圖，其顯示圓柱形微載體的頂面；2A and 2B are electron micrographs and schematic diagrams of microcarriers made according to a specific example of the present invention, which show the top surface of cylindrical microcarriers;

圖3A和3B分別為依據本發明一具體例所製成的微載體的另一電子顯微鏡照片和示意圖，其顯示圓柱形微載體的側面；3A and 3B are respectively another electron micrograph and a schematic diagram of a microcarrier made according to a specific example of the present invention, which shows the side of a cylindrical microcarrier;

圖4A和4B為依據本發明一具體例所製成的微載體的其他二個電子顯微鏡照片，其顯示微載體的孔洞結構；4A and 4B are other two electron micrographs of microcarriers made according to a specific example of the present invention, which show the hole structure of microcarriers;

圖5是依據本發明用於製造微載體的方法的流程圖；Fig. 5 is the flowchart of the method for making microcarrier according to the present invention;

圖6為依據本發明一具體例的光學顯微鏡照片，其顯示在模具中經過冷凍乾燥的泡沫體；以及Figure 6 is an optical micrograph showing freeze-dried foam in a mold according to an embodiment of the present invention; and

圖7為經過傳統切削工序所製成的微載體的電子顯微鏡照片。Fig. 7 is an electron micrograph of a microcarrier made through a conventional cutting process.

無none

100:微載體 100: Microcarriers

120:連續媒質 120: Continuous media

123:連續外壁 123: continuous outer wall

124:暴露孔 124: Exposure hole

Claims (14)

一種具有三維支架結構用於培養細胞之微載體，其包含： 一個由生物相容性高分子所構成的連續媒質，其具有一實質上呈現基本幾何體的構形，以及一介於500微米至3000微米的特徵尺寸； 其中該微載體形成有數個相互鄰接的球狀巨孔，而該等球狀巨孔之間透過一或多個連通孔相通，其中該等球狀巨孔的直徑與該特徵尺寸的比值介於約1：6至約1：10之間；以及 其中該微載體具有一連續外壁，而該等球狀巨孔與該連續外壁相接處分別形成有一暴露孔。 A microcarrier with a three-dimensional scaffold structure for culturing cells, comprising: a continuous medium of biocompatible polymers having a substantially elementary geometrical configuration and a characteristic size between 500 microns and 3000 microns; Wherein the microcarrier is formed with several spherical macropores adjacent to each other, and the spherical macropores communicate through one or more communicating holes, wherein the ratio of the diameter of the spherical macropores to the characteristic size is between between about 1:6 and about 1:10; and Wherein the microcarrier has a continuous outer wall, and an exposed hole is formed at the connection between the spherical macropores and the continuous outer wall. 如請求項1所述的微載體，其中該等暴露孔的孔徑實質小於與其相鄰的球狀巨孔的孔徑。The microcarrier according to claim 1, wherein the diameter of the exposed pores is substantially smaller than that of the adjacent spherical macropores. 如請求項2所述的微載體，其中該基本幾何體選自於由圓柱體、球體、圓錐體、正方體、長方體、稜柱體和稜錐體所組成的群組。The microcarrier according to claim 2, wherein the basic geometry is selected from the group consisting of cylinder, sphere, cone, cube, cuboid, prism and pyramid. 如請求項3所述的微載體，其中該連續媒質具有一介於500微米至880微米的特徵尺寸。The microcarrier of claim 3, wherein the continuous medium has a characteristic size between 500 microns and 880 microns. 如請求項4所述的微載體，其中該生物相容性高分子選自於由蛋白質類、多醣類、人造高分子類，以及它們之組合所組成的群組。The microcarrier according to claim 4, wherein the biocompatible macromolecule is selected from the group consisting of proteins, polysaccharides, artificial macromolecules, and combinations thereof. 如請求項5所述的微載體，其中該生物相容性高分子選自於由明膠、膠原蛋白、纖維蛋白(fibrins)、瓊脂糖、玻尿酸、甲殼素、海藻酸鹽、纖維素、吉蘭膠(gellan gum)所組成的群組。The microcarrier as described in claim item 5, wherein the biocompatible polymer is selected from gelatin, collagen, fibrin (fibrins), agarose, hyaluronic acid, chitin, alginate, cellulose, gellan A group consisting of gellan gum. 如請求項6所述的微載體，其中該等球狀巨孔具有介於50微米至200微米的直徑。The microcarrier according to claim 6, wherein the spherical macropores have a diameter between 50 microns and 200 microns. 如請求項7所述的微載體，其中該連續媒質中有至少50%的球狀巨孔是以最密堆積的形式排列。The microcarrier as claimed in item 7, wherein at least 50% of the spherical macropores in the continuous medium are arranged in the form of closest packing. 一種用於製造微載體的方法，其包含下列步驟： A、製備一個高分子泡沫體，其包含一連續相，以及一與該連續相不相混溶而且由許多個散佈於該連續相中的相互隔離單元構成的分散相，其中該連續相包含選自於由生物相容性高分子、其單體、其寡聚物和它們之組合所組成的群組的成份； B、將該高分子泡沫體填入一個多孔平板模具中，再將該高分子泡沫體固化而得到一連續媒質，其中該多孔平板模具界定出多個連接該多孔平板模具的兩個主要表面的微型通孔，且各個微型通孔被建構成具有基本幾何體的構形，並且具有一介於500微米至3000微米的特徵尺寸，以及其中該分散相中各個相互隔離單元具有的直徑與該特徵尺寸的比值介於約 1：6 至約 1：10；以及 C、使該連續媒質脫離該多孔平板模具，而得到該具有三維細胞支架和一連續外壁的微載體。 A method for producing microcarriers, comprising the steps of: A. Prepare a polymer foam, which includes a continuous phase, and a dispersed phase that is immiscible with the continuous phase and is composed of many mutually isolated units scattered in the continuous phase, wherein the continuous phase includes selected Components from the group consisting of biocompatible polymers, their monomers, their oligomers, and combinations thereof; B. Fill the polymer foam into a porous flat mold, and then solidify the polymer foam to obtain a continuous medium, wherein the porous flat mold defines a plurality of holes connecting the two main surfaces of the porous flat mold Micro through holes, and each micro through hole is constructed to have a basic geometric configuration, and has a characteristic size between 500 micrometers and 3000 micrometers, and wherein each isolated unit in the dispersed phase has a diameter corresponding to the characteristic size a ratio of about 1:6 to about 1:10; and C. Detaching the continuous medium from the porous plate mold to obtain the microcarrier with a three-dimensional cell scaffold and a continuous outer wall. 如請求項9所述用於製造微載體的方法，其中該基本幾何體選自於由圓柱體、球體、圓錐體、正方體、長方體、稜柱體和稜錐體所組成的群組。The method for manufacturing microcarriers as claimed in claim 9, wherein the basic geometry is selected from the group consisting of cylinder, sphere, cone, cube, cuboid, prism and pyramid. 如請求項10所述用於製造三維細胞支架的方法，其中該連續媒質具有一介於500微米至880微米的特徵尺寸。The method for fabricating a three-dimensional cell scaffold as claimed in claim 10, wherein the continuous medium has a characteristic size between 500 μm and 880 μm. 如請求項11所述用於製造微載體的方法，其中該生物相容性高分子選自於由蛋白質類、多醣類、人造高分子類，以及它們之組合所組成的群組。The method for manufacturing microcarriers as claimed in claim 11, wherein the biocompatible macromolecules are selected from the group consisting of proteins, polysaccharides, artificial macromolecules, and combinations thereof. 如請求項12所述用於製造微載體的方法，其中該生物相容性高分子選自於由明膠、膠原蛋白、纖維蛋白(fibrins)、瓊脂糖、玻尿酸、甲殼素、海藻酸鹽、纖維素、吉蘭膠(gellan gum)所組成的群組。The method for making microcarriers as described in claim 12, wherein the biocompatible polymer is selected from gelatin, collagen, fibrin (fibrins), agarose, hyaluronic acid, chitin, alginate, fiber The group consisting of plain, gellan gum (gellan gum). 如請求項13所述用於製造微載體的方法，其中該步驟A包含將一氣體導入該連續相中以產生氣泡，藉此使得該分散相是該氣體。The method for manufacturing microcarriers as claimed in claim 13, wherein the step A includes introducing a gas into the continuous phase to generate bubbles, thereby making the dispersed phase the gas.

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