Name: Isolation of Limbal Niche Cells (LNCs) from Limbus Tissue
Uploaded: 2023-10-27T14:10:00
Description: Watch this Scientific Journal Video about Isolation of Limbal Niche Cells LNCs from Limbus Tissue at JoVE.com

isolation of limbal niche cells (lncs) from limbus tissue

En protokoll for å isolere, dyrke og identifisere humane limbale nisjeceller

The incidence of corneal epithelial stem cell deficiency (CESD), also called limbal stem cells deficiency (LSCD)1, and&#160;corneal epithelial regeneration (CES) are&#160;becoming more and more urgent because of corneal infection&#160;and injury. If not properly treated, CESD can lead to blindness that requires corneal transplantation. As a result, CES regeneration is&#160;becoming more significant. There is a group of supportive cells called limbal niche cells (LNCs) that provide essential support for CES function. Limbal stromal stem cells were first isolated by Polisetty et al.2 and identified by Xie et al.3&#160;as LNCs which are localized in the limbal epithelium subjacent and stroma of the limbus. LNCs are the key supporting stem cell of the corneal rim&#160;and, with the function of bone marrow-derived MSC (BMMSCs), and could&#160;be induced to develop into corneal epithelial cells and corneal stromal cells, etc.3,4,5,6,7. Previous studies showed that the stem-cell qualities of LNCs are more primitive than BMMSCs8, which is already widely used in the clinic. LNCs may even become the next viable option after MSC, especially for treating CESD. As important supporting cells for CES, LNCs are also stem cells derived from the &#34;niche&#34; structure of the limbus. LNCs may play a key role in the dedifferentiation of mature corneal epithelial cells (MCEC) to CES9. However, studies on LNCs are still relatively insufficient, and there is no consensus on the terminology, isolation, purification, identification, and characteristics of LNCs. Some researchers have named LNCs limbal biopsy-derived stromal stem cells10, limbal mesenchymal stem cells11, limbal fibroblast stem cells12, and limbal mesenchymal stromal cells13. As the growth characteristics of LNCs have not been described in detail, and because of their promising scientific and clinical applications, and may be one of the most important clinical tools in the future, it is necessary to summarize the isolation, purification, identification, and characteristics of LNCs.
According to a previous study14, LNCs are mainly present at the limbal epithelium subjacent and stroma of the limbus. This protocol includes treating limbus tissue using collagenase A, obtaining a cluster consisting of LEPC and LNCs, and digesting it into single cells with 0.25% trypsin-EDTA (TE). LNCs were then selectively cultured in a modified embryonic stem cell medium (MESCM) to be purified. The protocol reported in this paper is simple and has high efficiency in obtaining human LNCs in large quantities.
The&#160;detailed procedure of LNC isolation, culture, and identification was recorded in the video for scientists who are interested in LNC&#160;study, and it can be conveniently repeated when needed.

Forfatter Spotlight: Standardisering av Limbal Niche Cell (LNC) isolasjon og karakterisering for å støtte utbredt LNC-forskning 

Isolering og identifisering av limbale nisjeceller

Our studies focus on the reconstruction of the limbal niche to treat limbal stem cell deficiency and to restore the patient's vision. We are trying to answer whether or not limbal niche cells are the key cells supporting the function of limbal stem cells, and if so, how it works. Recent studies reported that subconjunctival transplantation of mesenchymal stem cells from bone marrow prevented the limbal stem cell deficiency of alkali-burned eyes.One of the technologies currently used to advance our research is single cell sequencing, which can analyze the key signaling pathway activities at a single cell level for limbal niche cell characterizing. Although LNC have the potential to become an important clinical tool in the future, their growth characteristics have not been explored in much details. Considering their promising scientific and clinical applications, it is necessary to summarize the isolation, purification, identification, and characteristics of LNC.

Watch this Scientific Journal Video about Isolation of Limbal Niche Cells LNCs from Limbus Tissue at JoVE.com

Isolation of Limbal Niche Cells (LNCs) from Limbus Tissue  

Isolering av limbale nisjeceller (LNC) fra limbusvev

Utfør isolering av limbale nisjeceller, eller LNCer, mens du arbeider under sterile forhold på en ultraren arbeidsbenk. Hent limbusvevet fra mellomproduktet av hornhinnen. Bruk et sterilt kirurgisk rundt blad, skrap og fjern iris og endotel rundt hornhinnen.Neste, med det sterile kirurgiske runde bladet, kutt limbusvevet i 12 like stykker, og etterlater bare en millimeter vev på begge sider av hornhinnen og sclera. Deretter overfører du de 12 limbusvevstykkene til en 35 millimeter kulturplate, og tilsett 1 milliliter kollagenase A for å fordype vevet helt i platen. Fordøy vevet ved å inkubere kulturplaten i en 37 graders Celsius-celleinkubator i 18 timer.Tine matrigel eller kjellermembranmatrisen i kjøleskap satt til 4 grader Celsius. Forbered en sterilisert pose som inneholder 200 mikroliter og 1 milliliter tips, et 15 milliliter sentrifugerør og en 6-brønnplate, og legg posen ved minus 20 eller minus 80 grader Celsius i 20 minutter. Etter å ha hentet spissene, sentrifugerøret og 6 brønnplater fra kjøleren, fortsett å utføre de neste trinnene på en ultraren arbeidsbenk.Bruk en forkjølt spiss på 200 mikroliter, pipett 50 mikroliter av den tinte kjellermembranmatrisen i 1 milliliter MESCM, og bland den forsiktig. Ved hjelp av en forkjølt, 1 milliliters spiss montert på en pipette, overfør den resulterende 5% kjellermembranmatrisen til en enkelt brønn på den forkjølte 6-brønnkulturplaten. Rist forsiktig 6-brønnplaten horisontalt før du legger den i en inkubator ved 37 grader Celsius i omtrent en time for å forberede den 5% matrigelbelagte kulturplaten.Etter 18-timers fordøyelse, hente kollagenase A fordøyd limbal vev, hovedsakelig inneholder små synlige klynger. Arbeid under et stereomikroskop, bruk en 200 mikroliter pipettespiss for å løsne klyngene fra det ufordøyde sklerale vevet. Senk de frittliggende klyngene i 0,25 %Trypsin-EDTA i en 35 millimeter kulturplate, og inkuber platen i 15 minutter.Deretter tilsettes 1 milliliter MESCM med 10% knockout-serum til platen for å slukke cellefordøyelsen. Pipetter suspensjonen forsiktig opp og ned med en 1 milliliter pipettespiss for å bryte klyngene inn i individuelle celler. Overfør cellesuspensjonen til et 15 milliliter sentrifugerør, og sentrifuge den ved 200 G i fem minutter ved romtemperatur.Uten å forstyrre cellepelleten, fjern forsiktig supernatanten og tilsett 1 milliliter MESCM for å resuspendere cellene. Bruk en 1 milliliter pipettespiss til å pipette innholdet opp og ned to til tre ganger for å sikre at hele pelleten resuspenderes i MESCM. Samle 20 mikroliter fra suspensjonen for celletelling.Deretter overfører du cellesuspensjonen til 5% kjellermembranmatrise belagt 6 brønnplate ved hjelp av en 1 milliliter pipette. Legg MESCM for å gjøre det totale volumet i brønnen 2,5 milliliter. Rist forsiktig 6-brønnplaten horisontalt for å sikre en jevn fordeling av celler.Etter avbildning av cellene, overfør dem til en cellekulturinkubator ved 37 grader Celsius. Den demonstrerte protokollen fordøyde vellykket hornhinnen scleral randvev, og genererte klynger som kunne visualiseres under mikroskopet. Som forventet lignet klyngene formen på en larve.De primære LNCene vokste sakte og krevde omtrent 12 dager for kultur. LNC fra passasje 1 til passasje 8 trengte omtrent tre dager på å passere, men vekstraten for LNC etter passering 9 sank betydelig. Morfologisk var LNC spindelformet og rund i passasje 0.Etter passasje 3 var de spindelformede med ensartede størrelser. Antall celledoblinger eller NCD representerte vekstraten til LNCene. NCD og akkumulerende NCD-kurver viste at LNC-ene vokste raskest fra passasje 3 til passasje 5.

isolation-of-limbal-niche-cells-lncs-from-limbus-tissue

Research

JoVE Journal

Medicine

Isolation of Limbal Niche Cells (LNCs) from Limbus Tissue

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Here, we present a protocol to isolate and identify the human limbal niche cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

Perform the isolation of limbal niche cells, or LNCs, while working under sterile conditions on an ultra-clean workbench. Retrieve the limbus tissue from the intermediate term corneal storage medium. Using a sterile surgical round blade, scrape and remove the iris and endothelium around the cornea.Next, with the sterile surgical round blade, cut the limbus tissue into 12 equal pieces, leaving only one millimeter of tissue on both sides of the cornea and sclera. After that, transfer the 12 limbus tissue pieces to a 35 millimeter culture plate, and add 1 milliliter of collagenase A to completely immerse the tissues in the plate. Digest the tissues by incubating the culture plate in a 37 degree Celsius cell incubator For 18 hours.Thaw the matrigel or basement membrane matrix in a refrigerator set at 4 degrees Celsius. Prepare a sterilized bag containing 200 microliter and 1 milliliter tips, a 15 milliliter centrifuge tube, and a 6 well-plate, and place the bag at minus 20 or minus 80 degrees Celsius for 20 minutes. After retrieving the tips, centrifuge tube, and 6 well-plate from the chiller, proceed to perform the next steps on an ultra-clean workbench.Using a pre-chilled 200 microliter tip, pipette 50 microliters of the thawed basement membrane matrix into 1 milliliter of MESCM, and gently mix it. Using a pre-chilled, 1 milliliter tip fitted to a pipette, transfer the resultant 5%basement membrane matrix to a single well of the pre-chilled 6 well culture plate. Gently shake the 6 well-plate horizontally before placing it in an incubator at 37 degrees Celsius for about one hour to prepare the 5%matrigel-coated culture plate.Following the 18-hour digestion, retrieve the collagenase A digested limbal tissue, mostly containing small visible clusters. Working under a stereo microscope, use a 200 microliter pipette tip to detach the clusters from the undigested scleral tissues. Submerge the detached clusters in 0.25%Trypsin-EDTA in a 35 millimeter culture plate, and incubate the plate for 15 minutes.Then, add 1 milliliter of MESCM with 10%knockout serum to the plate to quench cell digestion. Gently pipette the suspension up and down with a 1 milliliter pipette tip to break the clusters into individual cells. Transfer the cell suspension to a 15 milliliter centrifuge tube, and centrifuge it at 200 G for five minutes at room temperature.Without disturbing the cell pellet, carefully remove the supernatant, and add 1 milliliter of MESCM to resuspend the cells. Using a 1 milliliter pipette tip, pipette the content up and down two to three times to ensure the entire pellet is resuspended in MESCM. Collect 20 microliters from the suspension for cell counting.Next, using a 1 milliliter pipette, transfer the cell suspension to the 5%basement membrane matrix coated 6 well-plate. Add MESCM to make the total volume in the well 2.5 milliliters. Gently shake the 6 well-plate horizontally to ensure a uniform distribution of cells.After imaging the cells, transfer them to a cell culture incubator at 37 degrees Celsius. The demonstrated protocol successfully digested the cornea scleral rim tissue, generating clusters that could be visualized under the microscope. As expected, the clusters resembled the shape of a caterpillar.The primary LNCs grew slowly and required approximately 12 days for culture. LNCs from passage 1 to passage 8 needed about three days to passage, but the growth rate of LNCs after passage 9 decreased significantly. Morphologically, LNCs were spindle-shaped and round in passage 0.After passage 3, they were spindle-shaped with uniform sizes. The number of cell doublings or NCD represented the growth rate of the LNCs. The NCD and accumulative NCD curves revealed that the LNCs grew the fastest from passage 3 to passage 5.

Identification of Limbal Niche Cells (LNCs)

To identify the limbal niche cells or LNCs by immunofluorescence staining, add 1 milliliter of 0.25%trypsin EDTA per well to the LNCs cultured in a 5%matrigel coated 6 well plate. Digest the cells by incubating the plate for five minutes at 37 degrees Celsius. Quench the digestion by adding 1 milliliter of knockout serum containing MESCM to the cell suspension.Transfer the cell suspension to a centrifuge tube and centrifuge it 200G for five minutes before carefully aspirating the supernatant. Re-suspend the pellet in one milliliter of MESCM. Next, using a cytofuge, deposit 80 microliters of the cell suspension equally on four microscope slides per the manufacturer's instruction.Fix the cells with 4%paraformaldehyde for approximately 10 minutes. Then permeabilize the cells on the slides using 0.2%Triton X-100 in PBS for 15 minutes. Block the cells with 2%bovine serum albumin for one hour before incubating them with primary antibodies on a shaker overnight at 4 degrees Celsius.Post incubation, remove unbound antibodies by washing the slides with PBST three times for five minutes each. Next, incubate the sample with suitable secondary antibodies at 37 degrees Celsius for one hour before washing any unbound antibodies as demonstrated previously. Counterstain the nuclei with DAPI having a concentration of 5 micrograms per milliliter.After sealing the slides, perform imaging using a fluorescence microscope. Using an RNA isolation kit extract the total RNA from the LNCs per the manufacturer's instructions. Then using a high capacity complementary DNA or CDNA transcription kit, reverse transcribe 1 to 2 micrograms of the RNA.Prepare a 20 microliter solution containing 2.5 microliters of the CDNA, 0.8 microliters of corresponding genetic primer, 10 microliters of a universal PCR master mix, and 6.7 microliters of double distilled water. Then perform a quantitative polymerase chain reaction using the appropriate conditions. Finally, employ the comparative CT technique to examine the relative gene expression.Double immuno staining of the fourth passage LNCs clearly revealed that these cells were consistently panCK negative, VIM positive, CD90 positive, CD105 positive, SCF positive, and PDGFR positive.