Abstract
BACKGROUND:Based on previous technique prepared for encapsulating living cells with alginate-polysine- alginate (APA) microcapsules, it has been confirmed that microencapsulated chromaffin cells have good analgesic effects. The immunoisolated effects of such microcapsule materials need to be evaluated.

OBJECTIVE: This study aimed to investigate the immunological rejections of APA microencapsulated chromaffin cells transplanted into rat anterior chamber of eyes and tendon of feet, and to evaluate the immunoisolated effect of microencapsulation.

DESIGN: A randomized controlled animal experiment.

SETTING: Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology.

MATERIALS: Forty-eight female SD rats, with the age of 3 months, were provided by the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology. The protocol was carried out in accordance with ethical guidelines for the use and care of animals. Alginate and polylysine used in the experiment were the products of Sigma Company, USA. Microcapsule generator was gifted by Germany.

METHODS: This study was performed at the Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology from September 2002 to September 2003. Suprarenal medulla was taken from 6 healthy adult cadavers of brain death. After isolated, digested and cultured, suprarenal medulla was prepared into chromaffin cell suspension. Written informed consents were obtained from the family members of donors, and the protocol was given approval by the Ethics Committee of the hospital. Empty microcapsules and microencapsulated cells were prepared by APA. The 48 rats were randomly divided into the human chromaffin cell (HCC) group, the empty microcapsule group and the microencapsulated HCC (ME-HCC) group. In each group, there were two transplanted regions of anterior chamber of eyes and tendon of feet, with 8 rats used for each region. Each rat in the HCC group was perfused 2×1010 L-1 cell suspension into the anterior chamber of eyes and tendon of feet. Those in the empty microcapsule group and the ME-HCC group were perfused 100 empty capsules and ME-HCCs (100 microcapsules, 400-500 HCCs per microcapsule) into the same regions, respectively.

MAIN OUTCOME MEASURES: On day 7 after transplantation, serum interleukin (IL)-2 level was determined by ELISA. Serum IgG and IgM levels were determined with a laser turbidimeter. On day 28 after transplantation, rat right eyeball and left feet were harvested, routinely sliced and stained by haematoxylin-eosin (HE). Histo-morphological structure was observed under a 40×light microscope.

RESULTS: Forty-eight rats were included in the final analysis. Serum IL-2, IgG and IgM levels were significantly lower in the empty microcapsule group and ME-HCC group than in the HCC group （t=8.544-21.64, P < 0.01）. A lot of lymphocyte and neutrophile infiltration could be found in the anterior chamber of eyes and tendon of feet of rats in the HCC group, but a little seen in that of the empty microcapsule group and ME-HCC group.

CONCLUSION: APA microencapsulation has an effective immunoisolated effect on immunological rejection due to its good biocompatibility and mechanical stability.

INTRODUCTION

Chromaffin cells can secrete catecholamine, enkephalin and many other neurotrophic active substances [1]. When transplanted into subarachnoid space, it can produce analgesic effect [2]. Central nervous system is an "immunologically privileged region", but some immunological rejections still exist in xenogenic cell transplantation and influence the long-time survival of xenograft. In comparison with central nervous system, anterior chamber of eyes and tendon of feet present no response to exotic transplanted tissue, called ocular "immune privilege" and foot "immune privilege". So anterior chamber of eyes and tendon of feet can substitute central nervous system in the study of immunological rejections, and they are easy to transplant, and easy to observe. The immunoisolated effect of microcapsule avoids the direct immune attack of recipient to microencapsulated chromaffin cell and prolongs their survival time in vivo [3]. In this study, we investigated the immunoissolated effect of microencapsulated human chromaffin cells (ME-HCC) cultured in vitro in the anterior chamber of eyes and tendon of feet, so as to provide evidence for xenogenic cell transplantation in clinical practice.

MATERIALS AND METHODS

Materials
This study was performed at the Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology from September 2002 to September 2003. Forty-eight female SD rats of clean grade, with the age of 3 months, weighing 180-250 g, were provided by the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology
[Permission No.SYXK (e)2004-0028]. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Nerve growth factors (NGF), cell culture medium (RPMI-1640), trypsase and fetal bovine serum were the products of Gibco Company, USA, and alginate and polylysine were the products of Sigma Company, USA. Microcapsule generator was gifted by Germany. CO2 incubator and two-phase inverted microscope were purchased from Sheldom Company.

Methods
Preparation of chromaffin cell suspension
Chromaffin cells were harvested from 6 healthy adult corpses of brain death, 5 males and 1 female. Written informed consents were obtained from the family members of donors, and the protocol was given approval by the Ethics Committee of the hospital. After isolated, digested, filtered and centrifuged, the suprarenal medulla of donors was cultured 3 days in RPMI 1640 culture medium supplemented with NGF and fetal bovine serum (0.2 in volume fraction). After cell morphological examination and tyrosine hydroxylase antibody-labeled immunohistochemical detection, the suprarenal medulla solution was prepared into cell suspension for later use [(2-4)×109-(2-4)×1011 L-1] [4].

Pre-transplantation treatment of chromaffin cells
Microencapsulated cells could be directly used in the transplantation, while non-microencapsulated HCC must be digested, diluted, centrifuged, re-suspended, counted and regulated at concentration before transplantation. Chromaffin cells were regulated to 5×1010 L-1 and 1×1010 L-1 before use. The former was used for HCC transplantation, and the latter for preparation of ME-HCCs. Before transplantation, chromaffin cells must be given the detection of bacterium, mycetes, mycoplasma and other pathogens. Only those with negative detection results could be used.

Preparation of empty microcapsules
Empty microcapsules prepared by alginate-polysine- alginate (APA) method of Sun et al [5] were temporarily preserved in the aseptic condition, and generally used within 1 hour.

Preparation and culture of ME-HCCs
Empty microcapsules with the diameter of 390-400 μm were prepared following Sun's APA method. They were rinsed three times with aseptic normal saline and then cultured at 37 ℃ in an incubator containing 5% CO2 air. Medium was renewed in the first 4-6 hours and then once every other day.

HCC transplantation
The 48 rats were randomly divided into the HCC group, the empty microcapsule group and the ME-HCC group. In each group, there were two transplanted regions at anterior chamber of eyes and tendon of feet, with 8 rats at each region. In the HCC group, each rat was intraperitoneally perfused 100 g/L chloral hydrate 0.35 g/kg, and its right eye was dripped 1% dicaine for ocular surface anesthesia. Thereafter, under the high-fold surgical microscope, anterior chamber was punctured via peripheral cornea with a thin needle for letting out a little aqueous fluid. Cell suspension (2×1010 L-1) was taken with a microinjector, and perfused into rat right ocular anterior chamber; Rats in the empty microcapsule group and the ME-HCC group were perfused 100 empty capsules and ME-HCCs (100 microcapsules, 400-500 HCCs per microcapsule) into the same regions with a one-off trochar（20#）, respectively.The rats for being perfused at the left tendon of feet were kept awake, and the other procedures were the same.

Determination of serum interleukin (IL)-2, immunoglobulin G( IgG) and immunoglobulin M (IgM) levels
On day 7 after transplantation, 2 mL blood was taken from rat-tail vein, and centrifuged for collected serum sample. Subsequently, it was preserved at -80 ℃ in the refrigerator for determining IL-2, IgG and IgM levels and observing their post-surgical changes. IL-2 was detected with an ELISA kit (Jingmei Company, Shenzhen, China) and an enzyme reader (DG 3022 A, made in China). Serum IgG and IgM levels were determined with a laser turbidimeter (Aray@, Backman Company). The reaction rate of single antibody for human with rat IgG and IgM was 15% and 35%, respectively [6]. The determined results were converted and then calculated.

Histomorphological observation of rat anterior chamber of eyes and tendon of feet
On day 28 after transplantation, the rats were sacrificed. Their right eyeballs and left feet were harvested for paraffin embedding. Eyeballs and feet were routinely sliced at sagittal plane and transverse plane, respectively. Thereafter, sections were stained with haematoxylin-eosin (HE) for histomorphological observation under a light microscope.

Statistical analysis
Statistical processing was carried out by the first author with SPSS 12.0 software. Data were expressed as Mean±SD. t test was used for intergroup comparison. A level of P < 0.05 was considered significant, and P < 0.01 was considered very significant.

RESULTS

Quantitative analysis of the experimental animals
Forty-eight rats were included in the final analysis, without deletion.

Comparison of serum IL-2, IgG and IgM levels of rats among the groups (Table 1)
Table 1 shows that serum IL-2, IgG and IgM levels were significantly lower in the empty microcapsule group and ME-HCC group than in the HCC group(P < 0.01).

Histomorphology of rat anterior chamber of eyes and tendon of feet
A lot of lymphocyte and neutrophile infiltration could be found in the anterior chamber of eyes and tendon of feet of rats in the HCC group, but a little seen in the empty microcapsule group and ME-HCC group.

DISCUSSION

Chromaffin cells transplanted into patients' subarachnoid space have analgesic effect, but immunological rejections of transplantation exist [7]. Immunological rejection is the biggest problem in the transplantation of tissues and cells, while immunoissolation can avoid the immunological rejections. Its mechanism is following: Some suitable materials are used to isolate grafts from host immune system, thus rejective reaction can be avoided, allowing grafts survive for a longer time and releasing bioactive molecules, such as hormone and neurotransmitters. Microcapsule is a widely used immunoisolation technique [8-10]. It allows nutrient substance, electrolyte, oxygen and other low-molecule substances (relative molecular weight < 30 000) pass through freely, but not large-molecule substance (relative molecular weight > 150 000), such as immunoglobulin, immunocyte and so on, and accordingly, rejective reactions can be avoided. Microcapsule can exert its biological effects by means of small molecule bioactive substance secreted by grafts.
Microencapsulation mainly focuses on microcapsule size and package materials. The diameter of microcapsule used in this study is 390-400 μm, which does not influence the survival and function of cells in the microcapsule[11]. Microcapsule materials are mainly to provide immune environment for grafts, therefore, the chemical components of coating materials directly influence the biocompatibility and physical stability of microcapsule [12-14]. APA microcapsule used in this study has very good biocompatibility and physical stability. It is demonstrated that APA microcapsule can effectively block natural killer cell-mediated cytotoxic effect, presuming that it can also effectively stop other cytotoxicity-related cells from entering into microcapsule, and effectively prevent the cells in the microcapsule from immunological rejection of host cells [15]. Polyglucosan molecules and IgG (relative molecular mass > 71 000) cannot pass through APA microcapsule [16]. Because IgG is the smallest immunoglobulin molecule, and the relative molecular mass of C3 component necessary for complement activation is 195 000, so microcapsule can effectively block the effects of cell immunity and humoral immunity on the cells in the microcapsule. In this study, we also detected IL-2 levels. Cytokines refer to the polypeptide-like factors synthesized and secreted by immunocytes and nonimmunocytes of body. They regulate the physiological function of many cells, and most of them have relative molecular mass less than 60000. People thought that cells which could secrete interferon-α, IL-1 and tumor necrosis factors gathered around the microcapsule and released such soluble factors, which entered into microcapsule and kill cells[17]. It was also found in the later days that IL-1 and IL-2, whose molecular mass was close to that of insulin, could not pass through APA coating.
In this study, we inoculated chromaffin cells into the regions in which immunological rejection is not easy to occur, such as anterior chamber of eyes and tendon of feet. Although anterior chamber of eyes and tendon of feet were thought immunologically privileged organ, some recent experiments have confirmed that they are not completely immunologically privileged, and cell transplantation there still causes immunological rejection [18]. In this study, we also found that the xenogenic transplantation of HCCs could cause the inflammatory reaction of anterior chamber of eyes and tendon of feet in recipients, presuming it is in relation to immunological rejection. So we used APA-coated chromaffin cells for the transplantation, in order to reduce the immunological rejection of xenogenic transplantation. Experimental results showed that ME-HCCs transplanted into anterior chamber of eyes and tendon of feet only cause slight immunological and inflammatory reactions. The immunological and inflammatory reactions were obviously milder in the empty microcapsule group and ME-HCC group than in the HCC group. We used microencapsulated HCCs in the experiments and found that serum IL-2, IgG and IgM levels of rats were lower in the ME-HCC group than in the HCC group. It indicates that microcapsule can effectively prevent IL-2, IgG, IgM and other cytokines and immune molecule substances from coming out of microcapsule, above-mentioned IL-2, IgG and IgM are reliable indexes for judging for acute immunological rejection, and reduction in immunological rejection is mainly caused by immunoissolation of microcapsule [19,20]. Our experiments confirmed that immunological rejections of xenogenic transplantation of ME-HCCs were markedly fewer than those of HCCs.
Our experimental results showed that microencapsulation could reduce the immunological rejections in the cell transplantation, and solve the problem of analgesia in the HCC transplantation, indicating that the xenogenic transplantation of ME-HCCs is a safe and promising method.

REFERENCES

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人嗜铬细胞微囊化处理对大鼠
异种移植的免疫隔离作用*☆

陈冀衡，杨茂元，李继勇，林献忠，毕好生
华中科技大学同济医学院附属同济医院麻醉学教研室，湖北省武汉市 430030
陈冀衡☆，女，1972年生，河北省唐县人，汉族，2004年华中科技大学同济医学院毕业，博士，主治医师，主要从事临床麻醉与疼痛机制的研究。现工作单位为北京肿瘤医院麻醉科。
国家自然科学基金课题资助项目（39900139）*
摘要
背景：课题组前期在建立海藻酸钠-多聚赖氨酸-海藻酸钠微囊包裹活细胞制备技术的基础上,已证明微囊化嗜铬细胞有良好的镇痛效果,而该微囊包被材料的免疫隔离作用尚需明确。
目的：观察海藻酸钠-多聚赖氨酸-海藻酸钠微囊化嗜铬细胞移植到大鼠眼前房和足胝部的免疫排斥反应，评价微囊化技术的免疫隔离作用。
设计：随机对照动物实验。
单位：华中科技大学同济医学院附属同济医院麻醉学教研室。
材料：选用雌性 SD大鼠48只，鼠龄3个月，由华中科技大学同济医学院实验动物部提供。实验过程中对动物处置符合动物伦理学标准。实验所用海藻酸钠、多聚赖氨酸为美国Sigma公司产品，微囊发生器为德国赠送。
方法：实验于2002-09/2003-09在华中科技大学同济医学院附属同济医院麻醉学实验室完成。①取6名脑死亡健康成人的肾上腺髓质，经分离、消化、培养后，制备成人嗜铬细胞悬液。供者家属对实验知情同意，实验方案通过医院伦理委员会批准。采用海藻酸钠-聚赖氨酸-海藻酸钠法制作空微囊和微囊化细胞。②48只大鼠被随机分为3组：人嗜铬细胞移植组、空微囊移植组、微囊化人嗜铬细胞移植组，每组分眼前房和足胝部两个部位进行移植，每个部位8只。人嗜铬细胞移植组分别将2×1010 L-1细胞悬液注入大鼠右眼前房和左足胝部。空微囊移植组和微囊化人嗜铬细胞组分别吸取空微囊（100个微囊）或ME-HCC（100个微囊，每个微囊包裹400~500个细胞）注入大鼠右眼前房和左足胝部。
主要观察指标：于移植术后第7天采用ELISA法测定血清白细胞介素2水平。采用激光散射比浊仪测定血清IgG和IgM水平。移植术后第28天取大鼠右侧眼球及左侧足组织作常规切片，苏木精-伊红染色，40倍光镜下观察组织形态。
结果：大鼠48只均进入结果分析。①血清白细胞介素2，IgG，IgM水平：空微囊移植组和微囊化人嗜铬细胞移植组均低于人嗜铬细胞移植组，差异有显著性意义（t=8.544~21.64,P < 0.01）。②大鼠眼前房和足胝部组织形态：人嗜铬细胞移植组大鼠的眼前房内和足胝部可见大量淋巴细胞和中性粒细胞浸润。空微囊移植组和微囊化人嗜铬细胞移植组大鼠眼前房和足胝部仅见少量淋巴细胞和中性粒细胞。
结论：海藻酸钠-多聚赖氨酸-海藻酸钠微囊化所产生的良好生物相容性及其机械稳定性，使之有效地发挥了免疫排斥隔离作用。
关键词：微囊化;嗜铬细胞;体外培养;免疫隔离;生物材料;组织工程
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)01-00166-04
陈冀衡,杨茂元,李继勇,林献忠,毕好生.人嗜铬细胞微囊化处理对大鼠异种移植的免疫隔离作用[J].中国组织工程研究与临床康复，2008，12(1):166-169
[www.zglckf.com/zglckf/ejournal/upfiles/08-1/1k-166(ps).pdf]
(Edited by Liu BL/Song LP/Wang L)