CN1560225A - Perfusion bioreactor system constructed in vitro with bilayer active skin - Google Patents
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Abstract
Description
技术领域technical field
本发明涉及一种生物反应器，具体地说涉及一种医学和组织工程学中体外构建双层活性皮肤的灌注式生物反应器。The invention relates to a bioreactor, in particular to a perfusion bioreactor for constructing double-layer active skin in vitro in medicine and tissue engineering.
背景技术Background technique
皮肤是人体最大的组织，其表面积约1.5-2.0m2。作为人体的最外一道屏障，皮肤也是最容易受到损伤的组织。每年大约有60000-80000烧伤病人需要进行皮肤移植。Skin is the largest tissue in the human body, with a surface area of about 1.5-2.0 m 2 . As the outermost barrier of the human body, the skin is also the most vulnerable tissue. Approximately 60,000-80,000 burn patients require skin grafts each year.
目前采用的自体皮肤移植、同种异体皮肤移植、异种皮肤移植存在供区不足、免疫排斥以及疾病传播等问题。组织工程皮肤的出现为皮肤移植注入了新的活力。Currently used autologous skin grafts, allogeneic skin grafts, and xenogeneic skin grafts have problems such as insufficient donor sites, immune rejection, and disease transmission. The emergence of tissue-engineered skin has injected new vitality into skin transplantation.
组织工程皮肤的研究始于二十世纪七十年代，至今已开发出了各种具有临床价值的皮肤替代物，按组成可以分为表皮替代物、真皮替代物和复合皮肤替代物。表皮替代物由于缺乏真皮层而存在薄而易碎、不耐磨、收缩较大等缺点，临床应用受到限制；没有表皮层的真皮替代物存在水分易蒸发易感染等缺点。理想的皮肤代用品应该能够将所缺失的真皮和表皮层同时修复，因为这两种成份不仅影响皮肤的功能和外形，而且会相互影响，促进彼此的分化。美国Organogenesis公司生产的Apligraf是第一种同时含有表皮和真皮的组织工程皮肤。The research on tissue-engineered skin began in the 1970s. So far, various skin substitutes with clinical value have been developed, which can be divided into epidermal substitutes, dermal substitutes and composite skin substitutes according to their composition. Due to the lack of dermis, epidermal substitutes have disadvantages such as thinness, fragility, non-wear resistance, and large shrinkage, which limit clinical application; dermal substitutes without epidermis have disadvantages such as easy evaporation of water and infection. An ideal skin substitute should be able to restore the missing dermis and epidermis at the same time, because these two components not only affect the function and appearance of the skin, but also affect each other and promote the differentiation of each other. Apligraf produced by American Organogenesis Company is the first tissue-engineered skin containing both epidermis and dermis.
组织工程皮肤构建的最终目的是进行皮肤移植为烧伤病人或者其它一些皮肤损伤(如外伤或手术引起的急性皮肤坏损、慢性溃疡等)患者提供暂时的或永久性的皮肤替代物。然而，一位典型的烧伤病人通常需要3000-5000cm2的皮肤替代物，为了能大规模生产皮肤替代物以满足临床需求，开发适用于体外构建人工皮肤的生物反应器系统是组织工程皮肤从实验室走向应用的必经之路。The ultimate goal of tissue engineered skin construction is to perform skin grafting to provide temporary or permanent skin substitutes for burn patients or other skin injuries (such as acute skin damage caused by trauma or surgery, chronic ulcers, etc.). However, a typical burn patient usually needs a skin substitute of 3000-5000cm 2 . In order to produce skin substitutes on a large scale to meet clinical needs, the development of a bioreactor system suitable for constructing artificial skin in vitro is an important aspect of tissue engineering skin from experiments. The only way for the laboratory to be applied.
Apligraf(William HE，Eaglastein MD，and Vincent Falanga，MD.Tissue engineeringand the development of Apligraf，a human skin equivalent.Clinical Therapeutics，1997，19(5)：894-905.)的生产是采用传统的静态培养，将成纤维细胞与胶原凝胶混合后铺于大小不一的培养皿，培养4-6天后在表面接上角质细胞，浸没培养2天后改用气液界面培养。这种手工制备方式操作复杂，不易放大，无法模拟体内的生长环境，且组织均一性、生产重复性和质量稳定性难以保证，因此近年来一些用于皮肤组织构建的生物反应器相继问世。Apligraf (William HE, Eaglastein MD, and Vincent Falanga, MD. Tissue engineering and the development of Apligraf, a human skin equivalent. Clinical Therapeutics, 1997, 19(5): 894-905.) is produced by traditional static culture, The fibroblasts are mixed with collagen gel and spread on culture dishes of different sizes. After 4-6 days of culture, keratinocytes are attached to the surface, and after 2 days of immersion culture, the air-liquid interface is used for culture. This manual preparation method is complicated to operate, difficult to scale up, unable to simulate the growth environment in the body, and it is difficult to guarantee tissue uniformity, production repeatability and quality stability. Therefore, some bioreactors for skin tissue construction have come out in recent years.
Halberstadt(Halberstadt CR，Hardin R，Bezverkov K，et al.The in vitro growthof a three-dimensional human dermal replacement using a single-pass perfusion system，Biotechnol Bioeng，1994，43：740-746.)等人设计开发了一种封闭的单向灌注培养系统，用于生产真皮组织替代物。该系统采用动态接种的方式，但培养系统结构复杂，且没有实现表皮层的构建。Prenosil和Villeneuve(PrenosilJE，Villeneuve PE.Automated production of cultured epidermal autograpts and sub-confluentepiderlmal autografts in a computer controlled bioreactor.Biotechnol Bioeng，1998，59：679-683.)开发了一套自动控制的表皮替代物反应器系统，该系统还是无法实现复合皮肤的生产。Halberstadt (Halberstadt CR, Hardin R, Bezverkov K, et al. The in vitro growth of a three-dimensional human dermal replacement using a single-pass perfusion system, Biotechnol Bioeng, 1994, 43: 740-746.) et al. designed and developed A closed, unidirectional perfusion culture system for the production of dermal tissue substitutes. The system adopts the method of dynamic inoculation, but the structure of the culture system is complicated, and the construction of the epidermis has not been realized. Prenosil and Villeneuve (PrenosilJE, Villeneuve PE. Automated production of cultured epidermal autografts and sub-confluent epidermal autografts in a computer controlled bioreactor. Biotechnol Bioeng, 1998, 59: 679-683.) developed a set of epidermal reactors for automatic control system, this system still cannot realize the production of composite skin.
Kermer(Michael Kremer，Ekkehard Lang，Alfred Berger.Organotypical engineeringof differentiated composited-skin equivalents of human keratinocytes in a collagen-GAGmatrix(INTEGRA Artificial Skin)in a perfusion culture system.Langenbeck’s Arch Surg，2001，386：357-363.)等人用MINUCELLS and MINUTISSUE开发的灌注系统用于复合皮肤的体外构建，然而他们直接将Integra人工皮肤作为真皮层，略去了真皮构建这一步，而且这种灌注装置在结构上没能支持气液界面，因此无法真正完成复合皮肤的体外构建。曹谊林等人在中国实用新型专利(申请号：02266979.5)中披露了一种组织工程皮肤体外构建的新型生物反应器，该反应器不仅结构复杂，而且对于支架材料内部细胞微环境的改善作用不大。Kermer (Michael Kremer, Ekkehard Lang, Alfred Berger. Organotypical engineering of differentiated composited-skin equivalents of human keratinocytes in a collagen-GAGmatrix (INTEGRA Artificial Skin) in a perfusion culture system. Langenbeck's3g3, 80-361) et al. used the perfusion system developed by MINUCELLS and MINUTISSUE for the in vitro construction of composite skin. However, they directly used the Integra artificial skin as the dermis layer, omitting the step of dermal construction, and this perfusion device was structurally unable to support gas-liquid interface, so the in vitro construction of composite skin cannot be truly completed. Cao Yilin and others disclosed a new type of bioreactor constructed in vitro for tissue engineered skin in the Chinese utility model patent (application number: 02266979.5). This reactor not only has a complex structure, but also has little effect on improving the cell microenvironment inside the scaffold material. .
发明内容Contents of the invention
本发明需要解决的技术问题是公开一种双层活性皮肤体外构建的灌注式生物反应器系统，以克服现有技术存在的结构复杂、无法模拟体内生长环境、不易放大的缺点，满足临床应用的需求。The technical problem to be solved in the present invention is to disclose a perfusion bioreactor system constructed in vitro with double-layer active skin, so as to overcome the shortcomings of the prior art that the structure is complex, the in vivo growth environment cannot be simulated, and it is not easy to scale up, so as to meet the requirements of clinical application. need.
本发明的技术构思是这样的：Technical concept of the present invention is such:
通过调节培养基的液面，在同一反应器中进行浸没培养或气液界面培养，以满足双层活性皮肤的制备工艺要求；通过对培养过程的监测，确定营养物消耗速率和代谢生成速率，及时反映细胞在生物反应器中生长情况，以确定合适的灌注速率并进行灌注培养，从而优化培养环境，满足细胞正常的生长和分泌胞外蛋白；通过液体分布器在细胞接种过程实现多点接种，使细胞在生物材料中分布更均匀，制备出的人工皮肤更接近天然皮肤的组织学结构和生理功能；本发明的生物反应器具有放大简单的特点，不仅可以放大单个反应器的尺寸，增加制备的人工皮肤的面积，还可由多个反应器并联，实现产品数量的增加，因此，可以从真正意义上实现体外大规模生产人工皮肤。By adjusting the liquid level of the medium, carry out immersion culture or air-liquid interface culture in the same reactor to meet the preparation process requirements of double-layer active skin; through monitoring the culture process, determine the nutrient consumption rate and metabolic production rate, Timely reflect the growth of cells in the bioreactor to determine the appropriate perfusion rate and perform perfusion culture, thereby optimizing the culture environment to meet the normal growth of cells and secretion of extracellular proteins; through the liquid distributor to achieve multi-point inoculation in the cell inoculation process , so that the cells are more evenly distributed in the biomaterial, and the prepared artificial skin is closer to the histological structure and physiological function of the natural skin; the bioreactor of the present invention has the characteristics of simple enlargement, not only can enlarge the size of a single reactor, but also increase The area of the prepared artificial skin can also be connected in parallel by multiple reactors to increase the number of products. Therefore, the large-scale production of artificial skin in vitro can be realized in a real sense.
本发明的双层活性皮肤体外构建的灌注式生物反应器系统至少包括：加液装置、灌注式生物反应器、收液装置、液体回流装置和供气装置；The perfusion bioreactor system constructed in vitro with double-layer active skin of the present invention at least includes: a liquid addition device, a perfusion bioreactor, a liquid collection device, a liquid return device and an air supply device;
所说的灌注式生物反应器包括：设有上盖的封闭容器；设置在封闭容器中将封闭容器分隔为上室和下室的多孔支撑板；设置在多孔支撑板上方靠近封闭容器内壁的紧固密封件；用于皮肤构建的支架材料或细胞材料复合物平放在多孔支撑板上，并通过紧固密封件固定，表皮层构建时多孔支撑板可提供气液界面；上室设有液体入口和液体出口，下室设有液体入口和液体出口，上盖设有液体入口、气体入口和气体出口，上盖下方设有液体分布器，用于分布从上盖1的液体入口进入的培养基。Said perfusion bioreactor comprises: a closed container provided with an upper cover; a porous support plate arranged in the closed container to divide the closed container into an upper chamber and a lower chamber; Solid seal; the scaffold material or cell material compound used for skin construction is placed flat on the porous support plate and fixed by the fastening seal. The porous support plate can provide an air-liquid interface when the epidermis is constructed; the upper chamber is equipped with a liquid Inlet and liquid outlet, the lower chamber is provided with a liquid inlet and a liquid outlet, the upper cover is provided with a liquid inlet, a gas inlet and a gas outlet, and a liquid distributor is provided under the upper cover for distributing the culture that enters from the liquid inlet of the upper cover 1 base.
所说的加液装置与灌注式生物反应器的液体进口相连接，用于将新鲜培养基输入灌注式生物反应器，所说的收液装置与灌注式生物反应器的液体出口相连接，用于收集反应器中反应后的培养基，所说的液体回流装置通过管线分别与加液装置和收液装置相连通，所说的供气装置通过管线与灌注式生物反应器的气体入口相连通；The liquid adding device is connected with the liquid inlet of the perfusion bioreactor, and is used for inputting fresh culture medium into the perfusion bioreactor, and the liquid receiving device is connected with the liquid outlet of the perfusion bioreactor for To collect the reacted culture medium in the reactor, the liquid return device is connected with the liquid feeding device and the liquid receiving device through pipelines, and the gas supply device is connected with the gas inlet of the perfusion bioreactor through pipelines ;
本发明的系统是这样使用的：The system of the present invention is used like this:
将用于皮肤构建的支架材料或细胞材料复合物平放在灌注式生物反应器内多孔支撑板上，并通过紧固密封件固定，新鲜培养基由加液装置送入灌注式生物反应器，通过液体分布器实现细胞多点接种，在反应器中进行浸没培养或气液界面培养，以满足双层活性皮肤的制备工艺要求，然后由灌注式生物反应器液体出口进入收液装置，空气供气装置送入灌注式生物反应器，以满足反应所需要的氧气和调节pH，液体回流装置可将收液装置收集的反应后的培养基送回加液装置，以再次使用。Put the scaffold material or cell material compound used for skin construction flat on the porous support plate in the perfusion bioreactor, and fix it by fastening the seal, and the fresh medium is sent into the perfusion bioreactor by the liquid feeding device, The multi-point inoculation of cells is realized through the liquid distributor, and the immersion culture or air-liquid interface culture is carried out in the reactor to meet the preparation process requirements of the double-layer active skin. Then the liquid outlet of the perfusion bioreactor enters the liquid collection device, and the air supply The gas device is sent to the perfusion bioreactor to meet the oxygen required for the reaction and adjust the pH. The liquid return device can send the reacted culture medium collected by the liquid collection device back to the liquid addition device for reuse.
本发明的反应器系统结构简单、易于放大，可进行湿热灭菌，能长时间维持无菌环境，能很好地模拟体内生长环境，实现从接种到真皮构建到表皮构建的整个双层活性皮肤体外构建过程。The reactor system of the present invention has a simple structure, is easy to scale up, can be sterilized by moist heat, can maintain a sterile environment for a long time, can well simulate the growth environment in the body, and realizes the entire double-layer active skin from inoculation to dermis construction to epidermis construction In vitro construction process.
附图说明Description of drawings
图1为双层活性皮肤体外构建的灌注式生物反应器系统整体结构组成示意图。Figure 1 is a schematic diagram of the overall structure and composition of a perfusion bioreactor system constructed in vitro with double-layer active skin.
图2为灌注式生物反应器主体的结构示意图。Fig. 2 is a schematic structural view of the main body of the perfusion bioreactor.
具体实施方式Detailed ways
参见图1和图2，本发明的双层活性皮肤体外构建的灌注式生物反应器系统至少包括：加液装置、灌注式生物反应器、收液装置、液体回流装置和供气装置；Referring to Fig. 1 and Fig. 2, the perfusion bioreactor system constructed in vitro with double-layer active skin of the present invention at least includes: a liquid addition device, a perfusion bioreactor, a liquid collection device, a liquid return device and an air supply device;
所说的灌注式生物反应器包括：设有上盖1的封闭容器2；设置在封闭容器2中将封闭容器2分隔为上室3和下室4的多孔支撑板5，设置在多孔支撑板5上方靠近封闭容器2内壁的紧固密封件6；用于皮肤构建的支架材料或细胞材料复合物7平放在多孔支撑板5上，并通过紧固密封件6固定，表皮层构建时多孔支撑板5可提供气液界面；上室3设有液体入口8和液体出口9，下室设有液体入口8和液体出口9，上盖1设有液体入口8、气体入口10和气体出口11，上盖1下方设有液体分布器101，用于分布从上盖1的液体入口8进入的培养基。Said perfusion bioreactor comprises: a closed container 2 provided with a loam cake 1; a porous support plate 5 arranged in the closed container 2 to separate the closed container 2 into an upper chamber 3 and a lower chamber 4, arranged on the porous support plate 5 above the fastening seal 6 close to the inner wall of the closed container 2; the scaffold material or cell material composite 7 for skin construction is placed flat on the porous support plate 5 and fixed by the fastening seal 6, and the epidermis is porous when it is constructed The support plate 5 can provide a gas-liquid interface; the upper chamber 3 is provided with a liquid inlet 8 and a liquid outlet 9, the lower chamber is provided with a liquid inlet 8 and a liquid outlet 9, and the upper cover 1 is provided with a liquid inlet 8, a gas inlet 10 and a gas outlet 11 , A liquid distributor 101 is provided under the upper cover 1 for distributing the culture medium entering from the liquid inlet 8 of the upper cover 1 .
所说的加液装置与灌注式生物反应器的液体入口8相连接，所说的收液装置与灌注式生物反应器的液体出口9相连接，所说的液体回流装置通过管线分别与加液装置和收液装置相连通，所说的供气装置通过管线与灌注式生物反应器的气体入口10相连通；The liquid feeding device is connected with the liquid inlet 8 of the perfusion bioreactor, the liquid receiving device is connected with the liquid outlet 9 of the perfusion bioreactor, and the liquid return device is respectively connected with the liquid feeding device through pipelines. The device is connected with the liquid receiving device, and said gas supply device is connected with the gas inlet 10 of the perfusion bioreactor through a pipeline;
由图1可见：It can be seen from Figure 1:
所说的加液装置包括加液蠕动泵12和与其相连接的加液储液瓶13，加液储液瓶13通过管线与灌注式生物反应器的液体入口8相连通，由于所说的生物反应器有三个液体入口8，接种时选用上盖1的液体入口8，灌注培养可能选择边侧的液体入口8，因此最好在加液蠕动泵12与灌注式生物反应器的各个液体入口8相连的管路之间设置控制阀门26，以控制液体从哪个入口8进入；Said liquid addition device comprises liquid addition peristaltic pump 12 and the liquid addition liquid storage bottle 13 that is connected with it, and liquid addition liquid storage bottle 13 is communicated with the liquid inlet 8 of perfusion bioreactor through pipeline, because said biological The reactor has three liquid inlets 8. The liquid inlet 8 of the upper cover 1 is used for inoculation, and the liquid inlet 8 on the side may be selected for perfusion culture. A control valve 26 is set between the connected pipelines to control which inlet 8 the liquid enters from;
所说的收液装置包括：收液蠕动泵14、收液储液瓶15、收液控制阀16和收液瓶17，收液蠕动泵14的进口与灌注式生物反应器的液体出口9相连通，收液蠕动泵14的出口与收液储液瓶15的入口相连通，收液储液瓶15的出口通过储液蠕动泵25与收液瓶17相连通，收液控制阀16设置在收液储液瓶15与储液蠕动泵25之间的管线上，用于控制收液量，生物反应器各个出口与收液蠕动泵14之间设有控制阀门26。Said liquid collection device comprises: liquid collection peristaltic pump 14, liquid collection storage bottle 15, liquid collection control valve 16 and liquid collection bottle 17, the inlet of liquid collection peristaltic pump 14 is connected with the liquid outlet 9 of perfusion bioreactor The outlet of the receiving liquid peristaltic pump 14 is connected with the inlet of the receiving liquid storage bottle 15, and the outlet of the receiving liquid storage bottle 15 is connected with the receiving liquid bottle 17 through the liquid storage peristaltic pump 25, and the liquid receiving control valve 16 is arranged on The pipeline between the liquid collection storage bottle 15 and the liquid storage peristaltic pump 25 is used to control the liquid collection volume, and a control valve 26 is provided between each outlet of the bioreactor and the liquid collection peristaltic pump 14 .
所说的液体回流装置包括通过回流管线19设置在收液储液瓶15与加液储液瓶13之间的回流控制阀18；Said liquid backflow device includes a backflow control valve 18 arranged between the liquid collection storage bottle 15 and the liquid addition storage bottle 13 through the return line 19;
所说的供气装置包括依次相互连接的空气泵20、空气流量计21和空气过滤器22，空气过滤器22与灌注式生物反应器相连接；Said air supply device comprises an air pump 20, an air flow meter 21 and an air filter 22 interconnected in sequence, and the air filter 22 is connected with the perfusion bioreactor;
所说的检测装置包括插在加液储液瓶13、收液储液瓶15中的溶氧电极23和收液储液瓶15中的pH电极24，溶氧电极23和pH电极24分别与溶氧测定仪和pH测定仪连接，以检测和控制加液储液瓶13、收液储液瓶15中培养基的溶氧浓度和pH。Said detection device comprises the dissolved oxygen electrode 23 inserted in the liquid storage bottle 13, the liquid storage bottle 15 and the pH electrode 24 in the liquid storage bottle 15, and the dissolved oxygen electrode 23 and the pH electrode 24 are respectively connected with The dissolved oxygen meter is connected with the pH meter to detect and control the dissolved oxygen concentration and pH of the culture medium in the liquid storage bottle 13 and the liquid storage bottle 15 .
由图1可见，可将多个所说的灌注式生物反应器并联连接，以满足扩大生产量的需要。It can be seen from Fig. 1 that a plurality of said perfusion bioreactors can be connected in parallel to meet the needs of expanding production capacity.
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