1、Chapter Four Microcapsulated Animal Cell Culture,第四章 动物细胞的微囊化培养,Section Microencapsulation of Animal Cell,Cultivation Methods of animal cellsMany cell lines, especially those derived from normal tissues, are considered to be Anchorage-Dependent, that is, they can only grow when attached to a suitabl
2、e substrate. Anchorage dependent cultureAnchorage dependent cell lines can be grown by the following methods:A - Conventional methods They include T flasks, Roux bottles and RollersB - ？,Fig 1 Schematic illustrating cell microencapsulation,INTRODUCTION,Microencapsulation is a process by which a cont
3、inuous film of polymeric material is coated on tiny droplets or particles of liquid or solid material. In other words, a microcapsule is a small sphere with a uniform surrounding wall. The material inside the microcapsule is called as the core, internal phase, or fill, while the wall is sometimes re
4、ferred to as a shell, coating or membrane (Kamyshny et al., 2006).Most of the microcapsules are very small spheres whose diameter ranges from a few micrometers to a few millimeters (Torrado et al., 2008). Bioencapsulation is the microencapsulation of a biologically active compound as a core material
5、 that is allowed to release at a certain rate (Socaciu, 2007).,Figure 2. Structure of Alginate Poly-lysine Alginate Microcapsule (Vos et al., 2008).,Figure 3 . Microcapsule with mean size of 732 m,Figure 4. Photomicrography of encapsulated RINm-5f cells after 7 days of cultivation,Fig 5. Microphotog
6、raphs of K562 cells entrapped in APA microcapsules,ARTIFICIAL CELLS,Life is endowed with a mysterious and divine life-force,Thomas Ming Swi Chang(born 1933, Shantou, China) is a Canadian physician and scientist. In 1957, while an undergraduate at McGill University he invented the worlds first artifi
7、cial cell.,Artificial Cells,Artificial microscopic structures Same size as biological cells Have some of the functional properties of biological cells.They contain biologically active materials.,Artificial cells,Like biological cells, Artificial Cells function with content retained inside to Act on
8、outside permanent moleculesRelease products of interaction,3. Protein Concentration and Buffer Exchange Using Ultrafiltration,Ultrafiltration (UF) is a pressure-driven membrane process used throughout downstream processing for: (1) protein concentration, (2) buffer exchange and desalting, (3) remova
9、l of small contaminants, (4) protein purification, and (5) virus clearance. This chapter will consider the first three applicationsother chapters in this volume discuss the final two processes.,Separation in UF is primarily owing to differences in solute size, with the larger species retained by the
10、 membrane whereas the solvent and smaller components pass into the filtrate through the membrane pores. Electrostatic (and other long-range) interactions can also affect the rate of solute transport, e.g., charged solutes are strongly excluded from the membrane pores during operation at low salt con
11、centrations.,UF membranes are cast from a wide range of polymers in both flat sheet and hollow fiber form. These membranes have an asymmetric structure with a very thin skin layer (approximately 0.5 m thick), which provides the membrane its selectivity, and a more macroporous substructure which prov
12、ides the required mechanical and structural integrity. UF membranes have mean pore size ranging from 10500.,However, most manufacturers rate their membranes by the nominal molecular weight cutoff, which is defined as the molecular weight of a solute with a particular retention coefficient (R): R = 1
13、 Cp/CFwhere Cp and CF are the solute concentrations in the permeate solution and feed stream, respectively.,Data are typically obtained with different model proteins or with polydisperse dextrans. Unfortunately, the procedures for assigning molecular weight cutoffs, including the choice of solutes,
14、the specific buffer and flow conditions, and the chosen retention value (e.g., R = 0.9) vary widely throughout the industry, making it difficult to use these classifications for actual process development.,Although small-scale UF processes can be performed using dead-end filtration, almost all large
15、-scale UF is performed using tangential flow filtration (TFF) in which the feed solution flows parallel to the membrane surface. A fraction of the feed is driven through the membrane by the imposed transmembrane pressure drop to form the filtrate or permeate, with the remaining solution collected as
16、 the retentate (see Fig. 1).,Fig. 1. Tangential flow filtration.,The tangential flow sweeps the surface of the membrane, reducing membrane fouling and increasing the filtrate flux (defined as the volumetric filtrate flow rate per unit membrane area). Typical filtrate flux in UF range from 25250 L m2
17、h1 (often written as LMH). Typical transmembrane pressures (TMP) in UF are 0.24 bar.,During UF, the retained biomolecules accumulate on the upstream surface of the membrane forming a concentration polarization layer. This layer reduces the effective pressure driving force and can provide an addition
18、al resistance to flow, both of which decrease the filtrate flux. In addition, the increase in solute concentration at the membrane surface increases the rate of solute transmission through the membrane.,At high TMP, the solute concentration at the membrane reaches a critical value, at which point th
19、e flux (J) becomes essentially independent of the transmembrane pressure. This critical concentration (Cw) may be related to the protein solubility or it may arise from osmotic pressure effects. A simple stagnant film model can be used to estimate the flux under these conditions : J = k ln(Cw/Cb) (2
20、),where Cw and Cb are the protein concentrations at the membrane surface and in the bulk solution, respectively. The mass transfer coefficient (k) characterizes the rate of back transport of solutes from the membrane surface. It is a function of device hydrodynamics (e.g., shear rate), solution prop
21、erties (viscosity and difffusion coefficient), and module geometry. The flux in the pressure-independent regime can be increased by increasing k (typically by increasing the tangential flow velocity) or by decreasing the bulk concentration of the retained species.,Ultrafiltration is generally perfor
22、med in batch mode as shown in the top panel of Fig. 2. The entire volume of feed is contained within a recycle tank. Protein concentration occurs by removal of filtrate through the membrane. Batch operations use a minimum of hardware, provide simple manual or automatic control, and provide the highe
23、st filtrate flux. However, it can be difficult to obtain very high concentration factors (large volume reduction) using batch operation, and it can also be difficult to maintain adequate mixing throughout the process. The fed batch configuration utilizes an additional tank to feed into the recycle t
24、ank (bottom panel of Fig. 2).,Fed batch processes can provide greater concentration factors than batch systems, and they also provide better mixing and increased flexibility for use in multiple processes. However, the fed batch configuration requires greater process time. In addition, the number of
25、passes through pumps and valves is much larger than in batch operation, and this can lead to increased protein denaturation and aggregation. Buffer exchange and desalting are accomplished using diafiltration (DF) in which the low molecular-weight components are washed away from the protein by simult
26、aneously adding fresh buffer (or solvent) to the feed during UF. Diafiltration is generally performed in batch mode, with the DF buffer added at a rate so as to maintain a constant retentate volume (top panel in Fig. 2).,Fig. 2. Ultrafiltration systems.,4. High-Performance Tangential Flow Filtration
27、 for Protein Separations,High-performance tangential flow filtration (HPTFF) is an emerging technology that uses semipermeable membranes for the separation of proteins without limit to their relative size . HPTFF can be used throughout the purification process to remove specific impurities (e.g., pr
28、oteins, DNA, or endotoxins), clear viruses, and/or eliminate protein oligomers or degradation products. In addition, HPTFF can effect simultaneous purification, concentration, and buffer exchange, providing the unique capability of combining several different separation steps into a single scalable
29、unit operation.,As originally described , HPTFF obtained high selectivity by careful control of filtrate flux and device fluid mechanics to minimize fouling and exploit the effects of concentration polarization (discussed subsequently). Effective separations in HPTFF are obtained by operating in the
30、 pressure-dependent, rather than the pressure-independent, regime. In addition, cocurrent flow on the filtrate side of the membrane could be used to maintain the optimal transmembrane pressure, and thus the maximum selectivity, throughout the module.,It was subsequently recognized that significant i
31、mprovements in performance could be obtained by controlling buffer pH and ionic strength to maximize differences in the effective volume of the different species. The effective volume of a charged protein (as determined by size exclusion chromatography) accounts for the presence of a diffuse electri
32、cal double layer surrounding the protein. Increasing the protein charge, or reducing the solution ionic strength, increases the effective volume thus reducing protein transmission through the membrane.,HPTFF can thus effect separations by exploiting differences in both size and charge, with the magn
33、itude of these contributions determined by the properties of the proteins as well as the choice of buffer conditions. Optimal performance is typically attained by operating close to the isoelectric point (pI) of the lower molecular weight protein and at relatively low salt concentrations (around 10
34、mM ionic strength) to maximize electrostatic interactions.,Even lower salt concentrations can be used, although there can be problems caused by pH shifts because of insufficient buffering capacity. Direct charge effects can be further exploited by using a membrane that has an electrical charge oppos
35、ite to that of the more highly retained protein. Note that it may be possible to exploit electrostatic interactions even for solutes with identical pI because of the different charge-pH profiles for the different species and the combined effects of protein charge and size on protein transmission thr
36、ough the membrane.,The feed flow in HPTFF is parallel to the membrane surface, with a fraction of the flow driven through the membrane by the applied transmembrane pressure drop to form the filtrate solution. This tangential flow “sweeps” the membrane surface, reducing the extent of fouling and incr
37、easing the filtrate flux (the volumetric filtrate flow rate per unit membrane area) compared to that obtained in dead-end systems.,Typical flux in HPTFF range from 15200 L m2h1 (often written as LMH). During HPTFF, the retained biomolecules accumulate at the upstream surface of the membrane. This ef
38、fect can be described by a simple stagnant film model: Cw = Cb exp(J/k),where Cw and Cb are the solute concentrations at the membrane surface and in the bulk solution, respectively, and J is the filtrate flux. The mass transfer coefficient (k) characterizes the rate of back transport of solutes from
39、 the membrane surface. It is a function of the module geometry, the fluid flow, and the solution properties (e.g., solution viscosity and solute diffusivity).,Concentration polarization has often been cited as an inherent limitation in using membrane systems for high resolution separations. However,
40、 proper choice of filtrate flux and mass transfer coefficient can be used to increase Cw, which increases the protein concentration in the permeate, resulting in significant improvements in overall system performance.,Protein separations in HPTFF are accomplished using a diafiltration mode in which
41、the impurity (or product) is washed out of the retentate by simultaneously adding fresh buffer to the feed reservoir as filtrate is removed through the membrane (see Fig. 1).,Fig. 1. HPTFF in the diafiltration mode.,This maintains an appropriate protein concentration in the retentate, minimizing mem
42、brane fouling and reducing protein aggregation/denaturation. Diafiltration is typically performed at constant retentate volume by controlling the rate of buffer addition to match the filtrate flow rate. Differential diafiltration can also be used, with the diafiltration and permeate flow rates adjus
43、ted to give the optimal concentration or dilution of the feed during HPTFF.,Fig. 2. HPTFF with permeate coflow.,Fig. 3. The use of cascades in HPTFF.,优点： 可防止细胞在培养过程中受到物理损伤. 活性蛋白不能从囊中自由出入半透膜，从而提高细胞密度和产物含量，并方便分离纯化处理.缺点： 微囊制作复杂，成功率不高. 微囊内死亡的细胞会污染正常产物. 收集产物必须破壁，不能实现生产连续化.,微囊化培养,微载体的直径在60-250m ，由天然葡聚糖、凝胶
44、或各种合成的聚合物组成，如聚苯乙烯、聚丙烯酰胺等。由这些材料及其改良型制成的微载体主要参考了细胞的粘附特性，在其表面带有大量电荷及其他生长基质物质，因而有利于细胞的粘附、铺展和增殖。,四、微囊化 微囊化培养技术其要点是：在无菌条件下将拟培养的细胞、生物活性物质及生长介质共同包裹在薄的半透膜中形成微囊，再将微囊放入培养系统内进行培养。生长介质为1.4%海藻酸钠溶液，半透膜由多聚赖氨酸形成。培养系统可采用搅拌式或气升式反应器系统。实验证实，采用批式和连续灌注式培养杂交瘤细胞生产单克隆抗体，在7-27 d 微囊内抗体浓度可达1250-5300 mg/ L。,利用微囊包裹具有特定功能的组织细胞，形成免
45、疫隔离的人工细胞，以此植入疾病动物或病人体内。1980 年报道了微囊化胰岛移植治疗糖尿病。他们将同种大鼠胰岛用海藻酸- 聚赖氨酸- 聚乙烯亚胺包埋后植入链脲霉素诱导的糖尿病大鼠体内，在未用免疫抑制剂的情况下，控制大鼠血糖正常达一年左右。,第一节 动物细胞的微囊化,微囊化是固定化技术中的一种，是用一层亲水性的半透膜将酶、辅酶、蛋白质等生物大分子或动植物细胞包围在珠状的微囊里，从而使得酶等生物大分子和细胞不能从微囊里逸出，而小分子的物质、培养基的营养物质可以自由出入半透膜，达到催化或培养的目的。,微囊化的神经组织细胞,一、微囊化概述,人工细胞的概念由加拿大马吉尔（McGill）大学T.M.S.Ch
46、ang教授于1957年首次提出并应用具有半渗透性薄膜的微胶囊固定活体细胞或组织取得成功。由于微胶囊膜起到了类似细胞膜的作用,且固定后体系的形态和功能酷似活性细胞,所以称之为“人工细胞”。,微囊化细胞的模式图,80 年代初, Lim 等人将微囊化技术与组织细胞移植相结合, 制备了具有良好生物相容性的海藻酸钠/聚赖氨酸(APA)微胶囊作为免疫隔离工具, 包埋猪胰岛细胞形成人工细胞, 并移植入糖尿病大鼠体内, 结果表明该人工细胞成功地调节了血糖水平, 代行了大鼠胰腺功能, 因而被称为人工胰腺. 这一研究成果较好地解决了组织细胞移植过程的免疫排斥问题, 避免或减少了昂贵的免疫抑制剂的使用, 为组织细胞
47、移植治疗神经/内分泌系统疾病提供了新思路.,二、动物细胞微囊化方法,需满足条件： 1、微囊化的过程要温和，快速，不损伤细胞，尽可能在液体状态和生理条件下制备。 2、微囊化所用的试剂和膜材料必须对细胞无毒害作用。 3、微囊化所形成的膜必须能够使营养物和代谢产物自由通过，膜的孔径可以控制。 4、膜应具有足够的机械强度以抵抗培养过程中的搅拌，不至于使微囊破裂,1、聚赖氨酸/海藻酸 (PLL/ALG) 微囊化 （1）原理 海藻酸是以1，4键连接的聚醛酸，其主要成分是甘露糖醛酸和古罗糖醛酸。 当它的水溶液以钙盐的形式存在时，则成凝胶状态。 用螯合剂将钙离子去除后，海藻酸又回复到溶液状态。 海藻酸钙凝胶用
48、聚赖氨酸处理后，其接触部分不再被螯合剂去钙而溶解,动物细胞与海藻酸溶液混合，经过微囊化发生器滴入CaCl2溶液中，形成凝胶微珠，然后用聚赖氨酸溶液处理微球表面，最后再用柠檬酸去除微珠的钙离子，这样，微珠内部的海藻酸成液态，动物细胞悬浮其中，而微珠表面由于受到聚赖氨酸的处理而不再溶解，形成一层薄膜，动物细胞就被这层膜包在微囊里了，形成细胞微囊。,（2）微囊化装置及步骤,制备微囊化动物细胞要经过以下几步（海藻酸-聚赖氨酸-海藻酸微囊） 1、无菌收集动物细胞，离心后再生理盐水洗涤。 2、离心收集细胞，并加入海藻酸溶液混合均匀，使成悬浮液。 3、将悬浮液装入微囊发生器中制成微滴。 4、微滴加到CaCl
49、2溶液中形成微胶珠。 5、凝胶珠用生理盐水洗去残留的CaCl2. 6、凝胶珠悬浮于聚赖氨酸溶液中，使之形成膜。 7、凝胶珠再用CaCl2溶液洗，用CHES缓冲液固化。 8、凝胶珠用生理盐水洗涤以后，再用ALG溶液处理。 9、生理盐水洗涤后的凝胶珠加0.05mg/l柠檬酸溶液处理，使半透膜内的ALG成液态，然后用生理盐水冲洗。,(3)影响微囊化的一些因素 微囊半透明孔径影响因素 微囊的质量影响因素 细胞的活性影响因素,微囊半透膜的孔径,是培养基营养成分、代谢物进行膜内外交换、截留一定分子量蛋白在囊内的关键，如何选择是一重要问题。 微囊的膜是多阴离子的海藻酸与多阳离子的聚赖氨酸相互作用而形成的。研
50、究表明，此半透膜的孔径主要由聚赖氨酸的分子量决定，一般来说用高分子量的聚赖氨酸(PLL)覆膜孔径大，PLL分子量低、孔径小，通常使用相对分子量为40000-80000的PLL。研究还表明，PLL溶液的浓度及处理时间、溶液的PH及使用温度也都会影响膜的孔径。,微囊的质量,海藻酸的纯度、黏度及甘露糖醛酸和古罗糖醛酸的比例都会影响微囊的形成及机械性能。纯度高、黏度高容易成囊。含古罗糖醛酸多的海藻酸容易成囊，机械性能好，不易破碎。因此在使用海藻酸时必须慎重地选择。,细胞的活性,在进行微囊化时要注意到保持动物细胞的存活率，这与微囊化时间的长短、温度、溶液的pH、保持溶液等渗等都有着密切的关系，需特别注意。微囊化时动物细胞在海藻酸溶液中的初始浓度也与存活率有关，不同的细胞，所用浓度不同，一般在104106个/ml,
