TWI407967B - 人類角膜內皮幹細胞之擴增方法 - Google Patents

人類角膜內皮幹細胞之擴增方法 Download PDF

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TWI407967B
TWI407967B TW096115095A TW96115095A TWI407967B TW I407967 B TWI407967 B TW I407967B TW 096115095 A TW096115095 A TW 096115095A TW 96115095 A TW96115095 A TW 96115095A TW I407967 B TWI407967 B TW I407967B
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Ray Jui Fang Tsai
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Description

人類角膜內皮幹細胞之擴增方法
本發明大體而言係關於一種擴增角膜內皮幹細胞之方法，更具體言之，本發明係關於一種例如角膜內皮重建時治療疑難的角膜內皮細胞失償(decompensation)之方法及移植物。
角膜內皮細胞係位於角膜後方之基底膜(即後彈力膜(Descemet’s membrane))上之單層扁平六角形細胞，其形成不含任何其他細胞類型之純細胞薄片。角膜內皮細胞對維持角膜之透明度十分重要。該功能係基於內皮對基質水合(stromal hydration)之調節，包括眼球水狀液之屏障及泵功能。眼內手術、青光眼、外傷或者先天性角膜疾病對人類角膜內皮造成之傷害可導致不可逆的角膜水腫，此至少部分係因為人類角膜內皮細胞在出生之後不具有絲分裂活性或具有極低之有絲分裂活性，以致細胞群體隨年齡增長而逐漸減少。
當對人類角膜內皮細胞之傷害引起角膜水腫時，通常建議使用同種異體移植物之後板層角膜移植術或穿透性角膜移植術治療。後板層角膜移植術或穿透性角膜移植術之一主要顧慮係一個捐贈者角膜只能提供一個病患之角膜內皮層移植。穿透性角膜移植術之另一顧慮係同種異體移植排斥反應。此外，在許多國家，捐贈者角膜的供應不足。
近來，有研究報導在兔子模型上，將裸露之羊膜用作培養人類角膜內皮細胞移植之載體(Ishino Y.等人之Invest Ophthalmol Vis Sci.2004；45：800-806)。然而，根據該等結果，該羊膜並未用作活體外(ex vivo)細胞擴增之基材。
本發明之一態樣係提供一種用於擴增人類角膜內皮幹細胞之方法，該方法包括：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或包括人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上；及c)於該羊膜上培養角膜內皮幹細胞一段足使該等角膜內皮幹細胞擴增至一適當面積之時間。
本發明之另一態樣提供一種產生用於病患植入部位之外科移植物之方法，該方法包括：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或含人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上；c)於該羊膜上培養角膜內皮幹細胞一段足使該等角膜內皮幹細胞擴增至一適當面積之時間；d)自該羊膜分離該等經培養之角膜內皮幹細胞；及e)將步驟d)中得到之該細胞移植至一載體上，以製得一外科移植物。
本發明之另一態樣係關於一種外科移植物，其可藉由本發明之上述方法製備。
本發明之再一態樣係關於一種治療病患缺乏角膜內皮細胞之損傷部位之方法，該方法包括：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或含人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上；c)於該羊膜上培養角膜內皮幹細胞一段足使該等角膜內皮幹細胞擴增至一適當面積之時間； d)自該羊膜分離該等經培養之角膜內皮幹細胞；e)將步驟d)中得到之細胞移植至一載體上，以製得一外科移植物；及f)將步驟e)中得到之該外科移植物移植於該損傷部位。
根據本發明的一實施例，提供一種用於擴增人類角膜內皮幹細胞之方法，該方法包括：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或含人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上；以及c)於該羊膜上培養角膜內皮幹細胞一段足使該等角膜內皮幹細胞擴增至一適當面積之時間。
根據本發明，羊膜係用於擴增人類角膜內皮幹細胞。該羊膜可含或不含羊膜細胞，其中該羊膜含細胞外間質。根據本發明之一實施例，在該羊膜之基底膜側進行活體外培養來擴增該角膜內皮幹細胞。將角膜內皮幹細胞置於該羊膜之基底膜側，使角膜內皮細胞相對於基底膜的位置為面向上方。於該羊膜上培養角膜內皮細胞一段足使該等角膜內皮幹細胞擴增至一適當面積或大小之時間，例如直徑約2至約3 cm之面積。
羊膜提供一種促進角膜內皮幹細胞群擴增的特殊合適的基材(niche substrate)。在本發明之實例中，於羊膜上擴增角膜內皮幹細胞後，自該羊膜分離該等角膜內皮幹細胞並將其移植至一載體，以製得一外科移植物。接著，將置於裸露之角膜後層表面上之所培養的角膜內皮幹細胞移植入病患眼睛。
本發明之另一態樣提供一種產生施用於病患移入部位 (recipient site)之外科移植物之方法，其包含下述步驟：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或含人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上；以及c)於該羊膜上培養角膜內皮幹細胞一段足使該等角膜內皮幹細胞擴增至一適當面積之時間；d)自該羊膜分離該等經培養之角膜內皮幹細胞；以及e)將步驟d)中得到之細胞移植至一載體上，以製得一外科移植物。
根據本發明之一實施例，步驟d)中使用之載體可為角膜圓片、經處理或改質之羊膜薄片或任一能承載細胞之適當基材。在本發明之一實施例中，載體可為大小介於約8.5 mm與約9.0 mm之間之角膜圓片。該角膜圓片的厚度約小於100μm。該角膜圓片可為取自捐贈者或眼庫(eye bank)之裸露角膜後層。在本發明之一實例中，該角膜盤為含有後彈力膜之角膜後層。
根據本發明之實施例，該等角膜內皮幹細胞係得自在對病患健康眼睛進行角膜後輪部組織切片(posterior limbal biopsy)內皮層，或取自捐贈者之內皮層之人類角膜內皮細胞，較佳為角膜內皮層周邊所取得之人類角膜內皮細胞。
本發明亦係關於一種用於病患缺乏角膜內皮細胞之損傷部位之外科移植物，其可藉由上述方法製備。
本發明也係關於一種治療病患缺乏角膜內皮細胞之損傷部位之方法，其中該方法包括：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或含人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上；以及c)於該羊膜上培養角膜內皮幹細胞一段足使該等角膜內皮幹 細胞擴增至一適當面積之時間；d)自該羊膜分離該等經培養之角膜內皮幹細胞；e)將步驟d)中取得之該等細胞移植至一載體上，以製得一外科移植物；及f)將步驟e)中取得之該外科移植物移植於該損傷部位。
參考以下特定、非限制性實例，進一步詳細描述本發明。
實例1：自角膜後輪部組織切片(limbal biopsy)製備人類角膜內皮幹細胞之培養
在進行小梁切除術(Trabeculectomy)期間，藉由活組織切片方式自病患或另一活體之相同眼或不同眼取得周邊之內皮層之一小片。使用優碘(Betadine.RTM.)(聚維酮碘(Prvidone-iodine))消毒眼瞼。在無菌條件下，距離輪部組織約5至6 mm之處，產生長度約8 mm以輪部組織為底之結膜瓣(conjunctival flap)。在距離輪部組織約3 mm處產生長度約5 mm、深度約400 μm以輪部組織為底之鞏膜瓣(scleral flap)，且該瓣延伸至角膜，並距離輪部組織約2 mm。當掀起鞏角膜瓣時，前房(anterior chamber)係由輪部組織區進入，且在該輪部組織區周圍平行延伸約3 mm的長度。進行小梁切除術，並自移除之組織分離角膜內皮層。接著使用分離的角膜內皮細胞進行培養，以10-0縫線間斷縫合鞏角膜瓣及結膜創口。
實例2：自捐贈者角膜製備人類角膜內皮幹細胞
自捐贈者角膜取得周邊角膜內皮層之小片。在進行穿透性角膜移植術(penetrating keratoplasty)時，環鋸中央角膜以取得捐贈者角膜之鞏角膜周緣(sclerocorneal rim)。使用第15號外科手術刀將具有後彈力層之角膜內皮層與小梁 組織網(Trabecular meshwork)仔細地分離。從一個捐贈者角膜取得六小片周邊角膜內皮層。
將含有角膜內皮幹細胞之周邊之角膜內皮層的小片置於含有1.5毫升(ml)培養基之35 mm培養皿中。该培養基含有OPTIMEM-1作為基礎培養基(basal medium)，補充5至8%病患血清、10奈克/毫升(ng/ml)之纖維母細胞生長因子(Fibroblast Growth Factor,FGF)、5 ng/ml之表皮生長因子(Epidermal Growth Factor,EGF)、10微克/毫升(μg/ml)之抗壞血酸、50 μg/ml之青黴素(penicilline)、50 μg/ml之鏈黴素(streptomycin)及2.5 mg/ml之防治黴(fungizone)。將培養皿中之小片立即送至實驗室培養。
實例3：製備人類羊膜及在該人類羊膜上擴增人類角膜內皮幹細胞
根據赫爾辛基宣言(Declaration of Helsinki)之宗旨並取得同意後，可在剖腹產時取得人類羊膜。使用含有抗生素之PBS(5 ml，含0.3%氧氟沙星(ofloxacin))清洗羊膜，然後存放於-80℃的DMEM及甘油中。
將羊膜基底膜側朝上，平滑地移於培養盤上，並在使用前置於37℃，含5%二氧化碳(CO2)及95%空氣的潮濕恆溫箱內培育過夜。於羊膜上進行角膜內皮外植體之培養。在含有約1至1.5 ml培養基之35 mm培養皿中，將含有角膜內皮幹細胞之周邊角膜內皮層植入/轉移至羊膜基底膜側。每隔兩天更換一次培養基，並培養2至4週，此時角膜內皮幹細胞已生長並擴散至直徑約2至3 cm之面積。藉由在37℃使用1.2國際單位(IU)裂解酶II(dispase Ⅱ)處理約1至2小時，接著再使用胰蛋白酶(trypsin)/乙二胺四乙酸(EDTA)處理數小時，將經培養之人類角膜內皮幹細胞自培養皿剝離，以取得供繼代培養用之細胞懸浮液。
實例4：製備人類角膜盤及將經擴增之人類角膜內皮 幹細胞移植至人類角膜圓片
在對患有大泡性角膜病變(bullous keratopathy)病患進行穿透性角膜移植術時，取得新鮮製備包括患病內皮細胞之全厚度的角膜中央圓片。如實例2所述，將全厚度的角膜中央圓片置於上述含有1.5 ml培養基之35 mm培養皿中。立即將培養皿中之角膜圓片送至實驗室。在漢克斯平衡鹽溶液(Hank’s Balanced Salt Solution(HBSS))中，與0.02 mg/ml胰蛋白酶/EDTA於37℃培育1小時以除去病患內皮細胞。藉由使用火焰拋光吸量管(flame-polished pipette)劇烈攪散可使細胞分開。以後彈力膜側朝上的方式將裸露之角膜圓片置於24孔之培養皿中，接著將實例3所取得之小體積(0.3至0.5 ml)之上述繼代培養用之細胞懸浮液，包含大約2×105個細胞，吸注至裸露角膜圓片之後彈力膜上，使用實例2所述之培養基培養約10至14天。細胞移植後，以逐漸減低之血清濃度(10%，5%，2%)，在37℃之培養基F99(哈姆氏F12/培養基199(Ham’s/Medium 199))中培育角膜約1星期。使用顯微鏡及掃描電子顯微鏡從形態學評估移植成功率。
參考圖1，放大倍率為1000倍之掃描電子顯微鏡影像顯示根據本發明所述方法之一實施例，將經活體外擴增之人類角膜內皮幹細胞移植至人類角膜圓片。人類角膜內皮之周邊區域就增殖及繼代培養而言，具有高度增殖能力。經活體外擴增之人類角膜內皮細胞之自體移植物(auto-graft)或同種異體移植物(allo-graft)及內皮角膜移植術為未來的角膜重建提供了最佳的解決辦法。
在預先處理過的羊膜基材上進行活體外擴增之獨特技術能確保健康及年輕的角膜內皮幹細胞之繼代培養並移植至接受者角膜。使用自體性角膜內皮幹細胞進行移植時，經擴增之人類角膜內皮幹細胞具有於移植後不需要施行免 疫抑制處理(immunosuppression)的優點。針對以此方式移植之同種異體角膜內皮幹細胞而言，因僅移植角膜內皮幹細胞而未移植其他類型細胞，減少排斥反應之發生率。
咸信對於熟習該項技術者而言，本文已充分揭示其獨特及具發明性之外科移植物及產生方法，且熟習該項技術者不需進行繁複的試驗即可實施本發明，且可在不脫離本發明之申請專利範圍所定義之精神及範圍下，對其作變更。
結合隨附之圖式將更容易理解本發明之上述發明內容及實施方式。為達成說明本發明之目的，圖中展示目前較佳的實施例。然而，應瞭解本發明並不限於圖式中展示之實施例。
圖式中：圖1係一掃描電子顯微鏡(SEM)影像，其顯示根據本發明之一實施例，在活體外擴增並移植至人類角膜圓片上之人類角膜內皮幹細胞。

Claims (11)

  1. 一種擴增人類角膜內皮幹細胞之方法，該方法包括：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或含人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上，其中該等人類角膜內皮幹細胞係得自健康眼睛的角膜後輪部組織切片(posterior limbal biopsy)內皮層或得自捐贈者的角膜後輪部組織切片內皮層；及c)於該羊膜上培養人類角膜內皮幹細胞一段足使該等人類角膜內皮幹細胞擴增至一適當面積之時間。
  2. 如申請專利範圍第1項之方法，其中於該羊膜之基底膜側上擴增該等人類角膜內皮幹細胞。
  3. 一種產生用於病患植入部位之外科移植物之方法，該方法包括：a)提供含或不含羊膜細胞之羊膜，其中該羊膜含細胞外間質；b)將一內皮層片或含人類角膜內皮幹細胞之細胞懸浮液置於該羊膜上，其中該等人類角膜內皮幹細胞係得自健康眼睛的角膜後輪部組織切片(posterior limbal biopsy)內皮層或得自捐贈者的角膜後輪部組織切片內皮層； c)於該羊膜上培養人類角膜內皮幹細胞一段足使該等人類角膜內皮幹細胞擴增至一適當面積之時間；d)自該羊膜分離該等經擴增之人類角膜內皮幹細胞；及e)將步驟d)中得到之該等細胞移植至一載體上，以製得一外科移植物。
  4. 如申請專利範圍第3項之方法，其中於該羊膜之一基底膜側上進行該等人類角膜內皮幹細胞之活體外擴增。
  5. 如申請專利範圍第3項之方法，其中該內皮層為一周邊之角膜內皮層。
  6. 如申請專利範圍第3項之方法，其中該載體為一角膜圓片或一羊膜薄片。
  7. 如申請專利範圍第3項之方法，其中該載體厚度小於約100 μm。
  8. 如申請專利範圍第6項之方法，其中該載體為一角膜圓片。
  9. 如申請專利範圍第8項之方法，其中該角膜圓片為一裸露之角膜後層。
  10. 如申請專利範圍第8項之方法，其中該角膜圓片為含有後彈力膜之一角膜後層。
  11. 一種如申請專利範圍第3-10項中任一項之方法製備之外科移植物。
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