CN101437939A - 人类角膜内皮细胞的扩增方法 - Google Patents

人类角膜内皮细胞的扩增方法 Download PDF

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CN101437939A
CN101437939A CNA2007800154559A CN200780015455A CN101437939A CN 101437939 A CN101437939 A CN 101437939A CN A2007800154559 A CNA2007800154559 A CN A2007800154559A CN 200780015455 A CN200780015455 A CN 200780015455A CN 101437939 A CN101437939 A CN 101437939A
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蔡瑞芳
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Cai Yaonian
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Abstract

一种扩增人类角膜内皮细胞的方法，其包括：a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；b)将内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；及c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间。本发明也关于一种使用上述扩增人类角膜内皮细胞的方法产生施用于病患植入部位(recipient site)的外科移植物的方法，及由该方法制备的外科移植物。

Description

人类角膜内皮细胞的扩增方法
技术领域
本发明大体而言涉及一种扩增角膜内皮细胞之方法，更具体言之，本发明涉及一种例如角膜内皮重建时治疗疑难的角膜内皮细胞失偿(decompensation)的方法及移植物。
背景技术
角膜内皮细胞位于角膜后方的基底膜(即后弹力膜(Descemet’s membrane))上的单层扁平六角形细胞，其形成不含任何其它细胞类型的纯细胞薄片。角膜内皮细胞对维持角膜的透明度十分重要。该功能是基于内皮对基质水合(stromalhydration)的调节，包括眼球水状液的屏障及泵功能。眼内手术、青光眼、外伤或者先天性角膜疾病对人类角膜内皮造成的伤害可导致不可逆的角膜水肿，此至少部分是因为人类角膜内皮细胞在出生之后不具有丝分裂活性或具有极低的有丝分裂活性，以致细胞群体随年龄增长而逐渐减少。
当对人类角膜内皮细胞之伤害引起角膜水肿时，通常建议使用同种异体移植物之后板层角膜移植术或穿透性角膜移植术治疗。后板层角膜移植术或穿透性角膜移植术的一个主要顾虑是一个捐赠者角膜只能提供一个病患的角膜内皮层移植。穿透性角膜移植术的另一顾虑是同种异体移植排斥反应。此外，在许多国家，捐赠者角膜的供应不足。
近来，有研究报导在兔子模型上，将裸露的羊膜用作培养人类角膜内皮细胞移植的载体(Ishino Y.等人的Invest Ophthalmol Vis Sci.2004；45:800-806)。然而，根据上述结果，该羊膜并未用作活体外(ex vivo)细胞扩增的基材。
发明内容
本发明的一个态样是提供一种用于扩增人类角膜内皮细胞的方法，该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或包括人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；及
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间。
本发明的另一态样提供一种产生用于病患植入部位的外科移植物的方法，该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间；
d)自该羊膜分离上述经培养的角膜内皮细胞；及
e)将步骤d)中得到的该细胞移植至一载体上，以制得外科移植物。
本发明的另一态样是关于一种外科移植物，其可通过本发明的上述方法制备。
本发明的再一态样是关于一种治疗病患缺乏角膜内皮细胞的损伤部位的方法，该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间；
d)自该羊膜分离上述经培养的角膜内皮细胞；
e)将步骤d)中得到的细胞移植至一载体上，以制得外科移植物；及
f)将步骤e)中得到的该外科移植物移植于该损伤部位。
附图说明
结合附图将更容易理解本发明的上述发明内容及实施方式。为达成说明本发明的目的，图中展示目前较佳的实施例。然而，应了解本发明并不限于附图中展示的实施例。
附图中：
图1是扫描电子显微镜(SEM)影像，其显示根据本发明一实施例，在活体外扩增并移植至人类角膜圆片上的人类角膜内皮干细胞。
具体实施方式
根据本发明的一实施例，提供一种用于扩增人类角膜内皮细胞的方法，该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；以及
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间。
根据本发明，羊膜用于扩增人类角膜内皮细胞。该羊膜可含或不含羊膜细胞，其中该羊膜含细胞外间质。根据本发明之一实施例，在该羊膜的基底膜侧进行活体外培养来扩增该角膜内皮干细胞。将角膜内皮干细胞置于该羊膜的基底膜侧，使角膜内皮细胞相对于基底膜的位置为面向上方。于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积或大小之时间，例如直径约2至约3cm的面积。
羊膜提供一种促进角膜内皮干细胞群扩增的特殊合适的基材(nichesubstrate)。在本发明的实例中，于羊膜上扩增角膜内皮细胞后，自该羊膜分离该等角膜内皮细胞并将其移植至一载体，以制得外科移植物。接着，将置于裸露的角膜后层表面上的所培养的角膜内皮细胞移植入病患眼睛。
本发明的另一态样提供一种产生施用于病患移入部位(recipient site)的外科移植物的方法，其包含下述步骤：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；以及
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间；
d)自该羊膜分离上述经培养的角膜内皮细胞；以及
e)将步骤d)中得到的细胞移植至一载体上，以制得外科移植物。
根据本发明一实施例，步骤d)中使用的载体可为角膜圆片、经处理或改质的羊膜薄片或任一能承载细胞的适当基材。在本发明一实施例中，载体可为大小介于约8.5mm与约9.0mm之间的角膜圆片。该角膜圆片的厚度约小于100μm。该角膜圆片可为取自捐赠者或眼库(eye bank)的裸露角膜后层。在本发明一实例中，该角膜盘为含有后弹力膜的角膜后层。
根据本发明的实施例，上述角膜内皮干细胞得自在对病患健康眼睛进行角膜后轮部组织切片(posterior limbal biopsy)内皮层，或取自捐赠者的内皮层的人类角膜内皮细胞，较佳为角膜内皮层周边所取得的人类角膜内皮细胞。
本发明亦关于一种用于病患缺乏角膜内皮细胞的损伤部位的外科移植物，其可通过上述方法制备。
本发明也关于一种治疗病患缺乏角膜内皮细胞的损伤部位的方法，其中该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；以及
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间；
d)自该羊膜分离上述经培养的角膜内皮细胞；
e)将步骤d)中取得的上述细胞移植至一载体上，以制得外科移植物；及
f)将步骤e)中取得的该外科移植物移植于该损伤部位。
参考以下特定、非限制性实例，进一步详细描述本发明。
实例1：自角膜后轮部组织切片(limbal biopsy)制备人类角膜内皮干细胞的培养
在进行小梁切除术(Trabeculectomy)期间，通过活组织切片方式自病患或另一活体的相同眼或不同眼取得周边的内皮层的一小片。使用优碘(Betadine.RTM.)(聚维酮碘(Prvidone-iodine))消毒眼睑。在无菌条件下，距离轮部组织约5至6mm之处，产生长度约8mm以轮部组织为底之结膜瓣(conjunctivalflap)。在距离轮部组织约3mm处产生长度约5mm、深度约400μm以轮部组织为底之巩膜瓣(scleral flap)，且该瓣延伸至角膜，并距离轮部组织约2mm。当掀起巩角膜瓣时，前房(anterior chamber)由轮部组织区进入，且在该轮部组织区周围平行延伸约3mm的长度。进行小梁切除术，并自移除的组织分离角膜内皮层。接着使用分离的角膜内皮细胞进行培养，以10-0缝线间断缝合巩角膜瓣及结膜创口。
实例2：自捐赠者角膜制备人类角膜内皮干细胞
自捐赠者角膜取得周边角膜内皮层的小片。在进行穿透性角膜移植术(penetrating keratoplasty)时，环锯中央角膜以取得捐赠者角膜的巩角膜周缘(sclerocorneal rim)。使用第15号外科手术刀将具有后弹力层的角膜内皮层与小梁组织网(Trabecular meshwork)仔细地分离。从一个捐赠者角膜取得六小片周边角膜内皮层。
将含有角膜内皮干细胞的周边的角膜内皮层的小片置于含有1.5毫升(ml)培养基的35mm培养皿中。该培养基含有OPTIMEM-1作为基础培养基(basalmedium)，补充5至8％病患血清、10纳克/毫升(ng/ml)的纤维母细胞生长因子(Fibroblast Growth Factor，FGF)、5ng/ml的表皮生长因子(Epidermal GrowthFactor，EGF)、10微克/毫升(μg/ml)的抗坏血酸、50μg/ml的青霉素(penicilline)、50μg/ml的链霉素(streptomycin)及2.5mg/ml的防治霉(fungizone)。将培养皿中的小片立即送至实验室的无菌层流净化罩中培养。
实例3：制备人类羊膜及在该人类羊膜上扩增人类角膜内皮干细胞
根据赫尔辛基宣言(Declaration of Helsinki)的宗旨并取得同意后，可在剖腹产时取得人类羊膜。使用含有抗生素的PBS(5ml，含0.3％氧氟沙星(ofloxacin))清洗羊膜，然后存放于-80℃的DMEM及甘油中。
将羊膜基底膜侧朝上，平滑地移于培养盘上，并在使用前置于37℃，含5％二氧化碳(CO2)及95％空气的潮湿恒温箱内培育过夜。于羊膜上进行角膜内皮外植体的培养。在含有约1至1.5ml培养基的35mm培养皿中，将含有角膜内皮干细胞的周边角膜内皮层植入/转移至羊膜基底膜侧。每隔两天更换一次培养基，并培养2至4周，此时角膜内皮干细胞已生长并扩散至直径约2至3cm的面积。通过在37℃使用1.2国际单位(IU)裂解酶II(dispase II)处理约1至2小时，接着再使用胰蛋白酶(trypsin)/乙二胺四乙酸(EDTA)处理数小时，将经培养的人类角膜内皮干细胞自培养皿剥离，以取得供继代培养用的细胞悬浮液。
实例4：制备人类角膜盘及将经扩增的人类角膜内皮干细胞移植至人类角膜圆片
在对患有大泡性角膜病变(bullous keratopathy)病患进行穿透性角膜移植术时，取得新鲜制备包括患病内皮细胞的全厚度的角膜中央圆片。如实例2所述，将全厚度的角膜中央圆片置于上述含有1.5ml培养基的35mm培养皿中。立即将培养皿中的角膜圆片送至实验室的无菌层流净化罩中培养。在汉克斯平衡盐溶液(Hank’s Balanced Salt Solution(HBSS))中，与0.02mg/ml胰蛋白酶/EDTA于37℃培育1小时以除去病患内皮细胞。通过使用火焰抛光吸量管(flame-polishedpipette)剧烈搅散可使细胞分开。以后弹力膜侧朝上的方式将裸露的角膜圆片置于24孔的培养皿中，接着将实例3所取得的小体积(0.3至0.5ml)的上述继代培养用的细胞悬浮液，包含大约2×105个细胞，吸注至裸露角膜圆片的后弹力膜上，使用实例2所述的培养基培养约10至14天。细胞移植后，以逐渐减低的血清浓度(10％，5％，2％)，在37℃的培养基F99(哈姆氏F12/培养基199(Ham’s/Medium199))中培育角膜约1星期。使用显微镜及扫描电子显微镜从形态学评估移植成功率。
参照图1，放大倍率为1000倍的扫描电子显微镜影像显示根据本发明所述方法的一实施例，将经活体外扩增的人类角膜内皮干细胞移植至人类角膜圆片。人类角膜内皮的周边区域就增殖及继代培养而言，具有高度增殖能力。经活体外扩增的人类角膜内皮细胞的自体移植物(auto-graft)或同种异体移植物(allo-graft)及内皮角膜移植术为未来的角膜重建提供了最佳的解决办法。
在预先处理过的羊膜基材上进行活体外扩增的独特技术能确保健康及年轻的角膜内皮干细胞的继代培养并移植至接受者角膜。使用自体性角膜内皮干细胞进行移植时，经扩增的人类角膜内皮干细胞具有于移植后不需要施行免疫抑制处理(immunosuppression)的优点。针对以此方式移植的同种异体角膜内皮干细胞而言，因仅移植角膜内皮干细胞而未移植其它类型细胞，减少排斥反应的发生率。
相信对于所属技术领域的技术人员而言，本文已充分揭示其独特及具发明性的外科移植物及产生方法，且所属技术领域的技术人员不需进行繁复的试验即可实施本发明，且可在不脱离本发明的权利要求所定义的精神及范围下，对其作变更。

Claims (23)

1.一种扩增人类角膜内皮细胞的方法，该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；及
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间。
2.根据权利要求1所述的方法，其特征在于于该羊膜的基底膜侧上扩增上述角膜内皮干细胞。
3.一种产生用于病患植入部位的外科移植物的方法，该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将内皮层片或含人类角膜内皮干细胞之细胞悬浮液置于该羊膜上；及
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间；
d)自该羊膜分离该等经培养的角膜内皮细胞；及
e)将步骤d)中得到之该等细胞移植至一载体上，以制得外科移植物。
4.根据权利要求3所述的方法，其特征在于于该羊膜的基底膜侧上进行上述角膜内皮干细胞之的活体外培养。
5.根据权利要求3或4所述的方法，其特征在于上述角膜内皮干细胞取自于在该病患的健康眼睛进行后轮部组织切片的角膜内皮层。
6.根据权利要求3或4所述的方法，其特征在于上述角膜内皮干细胞取自于捐赠者的内皮层。
7.根据权利要求6所述的方法，其特征在于该内皮层为一周边的角膜内皮层。
8.根据权利要求3至7中任一项所述的方法，其特征在于该载体为角膜圆片或羊膜层片。
9.根据权利要求3至8中任一项所述的方法，其特征在于该载体厚度小于约100μm。
10.根据权利要求8所述的方法，其特征在于该载体为角膜圆片。
11.根据权利要求10所述的方法，其特征在于该角膜圆片为裸露的角膜后层。
12.根据权利要求10所述的方法，其特征在于该角膜圆片为含有后弹力膜的角膜后层。
13.一种根据权利要求3-12中任一项所述的方法制备的外科移植物。
14.一种治疗病患缺乏角膜内皮细胞的损伤部位的方法，该方法包括：
a)提供含或不含羊膜细胞的羊膜，其中该羊膜含细胞外间质；
b)将一内皮层片或含人类角膜内皮干细胞的细胞悬浮液置于该羊膜上；及
c)于该羊膜上培养角膜内皮细胞一段足使上述角膜内皮干细胞扩增至一适当面积的时间；
d)自该羊膜分离上述经培养的角膜内皮细胞；及
e)将步骤d)中得到的上述细胞移植至一载体上，以制得外科移植物；及
f)将步骤e)中得到的该外科移植物移植于该损伤部位。
15.根据权利要求14所述的方法，其特征在于于该羊膜的基底膜侧上进行上述角膜内皮干细胞的活体外培养。
16.根据权利要求14或15所述的方法，其特征在于上述角膜内皮干细胞取自于在该病患的健康眼睛进行后轮部组织切片的角膜内皮层。
17.根据权利要求14或15所述的方法，其特征在于上述角膜内皮干细胞取自于捐赠者的内皮层。
18.根据权利要求14至17中任一项的方法，其特征在于该内皮层为一周边的角膜内皮层。
19.根据权利要求14至18中任一项所述的方法，其特征在于该载体为角膜圆片或羊膜层片。
20.根据权利要求14至19中任一项所述的方法，其特征在于该载体厚度小于约100μm。
21.根据权利要求14至20中任一项所述的方法，其特征在于该载体为角膜圆片。
22.根据权利要求21所述的方法，其特征在于该角膜圆片为裸露的角膜后层。
23.根据权利要求21所述的方法，其特征在于该角膜圆片为含有后弹力膜的角膜后层。
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CN104837494A (zh) * 2012-09-05 2015-08-12 亚洲大学校产学协力团 使用软骨来源细胞的胞外基质膜的治疗血管生成疾病的组合物、以及角膜或结膜的移植物

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