이 원고는 미국 국립보건원(National Institutes of Health)의 R01DK118568(MG), R01DK124614(MG) 및 R01HD105301(MG), Chan Zuckerberg Initiative Grant 2022-316749(MG), Thrasher Research Fund Early Career Award(LCF), UNC Children's Development Early Career Investigator Grant(LCF)의 지원을 받았습니다. 채플 힐에 있는 노스캐롤라이나 대학교의 소아과.

엔테로이드는 NEC 또는 비염증성 병인이 있는 기타 장 질환에 대한 수술 시 얻은 미숙아(임신 22주에서 36주에 출생)의 소장 샘플에서 유래되었습니다. 모든 검체 채취 및 처리는 세인트루이스에 있는 워싱턴 대학교(IRB 프로토콜 번호 201706182 및 201804040)와 채플힐에 있는 노스캐롤라이나 대학교(IRB 프로토콜 번호 21-3134)의 기관 검토 위원회(Institutional Review Boards)의 정보에 입각한 동의 및 승인을 받은 ...

기계론적 연구 및 약물 발견을 위한 괴사성 장염 모델

<ol>
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이 NEC-on-a-chip 시스템은 NEC의 병태생리학을 모델링하는 데 사용할 수 있는 강력한 새 도구입니다. 이 플랫폼은 연속적인 발광 흐름 및 스트레치가 있는 공동 배양 시스템을 통합하여 이전 모델보다 생체 내 장 내 환경과 더 유사한 복잡한 미세환경을 제공합니다. 이러한 조건은 성숙한 상피 하위 유형과 단단한 접합부로 구성된 고도로 편광된 상피가 늘어선 3D 융모와 같은 구조의 발달을 촉?...

괴사성 장염(Necrotizing enterocolitis, NEC)은 조산아에게 영향을 미치며, 체중 1500g < 출생아의 경우 발병률이 최대 10%에 이른다1. NEC의 병태생리학은 복잡하며 장 상피의 손상, 장 밀착 접합부의 파괴, 장 장벽 투과성 증가, 면역 조절 장애 및 상피 세포 사멸을 포함합니다 2,3. NEC의 발병기전과 관련된 메커니즘에 대한 우리의 이해는 불완전하며, 수십 년간의 연구에도 불구하고 효과적인 표적 치료법은 아직 없습니다.
NEC 연구의 진전을 가로막는 중요한 장벽은 인간 유아로부터 분리한 1차 장 조직의 가용성이 제한적이고 크기가 작다는 것입니다. NEC를 앓고 있는 영아의 장 조직을 절제하면 괴사하고 심하게 손상되는 경우가 많아 질병 발병 전 메커니즘에 대한 연구가 복잡해집니다. 예를 들어, NEC를 앓고 있는 영아의 소장에는 면역 세포가 넘쳐나며, 장 줄기세포의 수가 감소하고, 상피 세포 증식이 감소하며, 상피 세포 사멸이 증가한다 4,5,6,7. 이로 인해 이러한 샘플에서 장 상피 세포를 배양하고 적대적인 염증 환경에서 분해될 수 있는 RNA와 단백질을 분리하는 데 어려움이 있습니다. 또한, 외과적 NEC를 앓고 있는 영아의 경우 질병 진행이 이미 진행되었기 때문에 질병을 유발하는 요인에 대한 기계론적 연구는 실현 불가능합니다. 이러한 한계로 인해 NEC의 기계론적 연구를 위해 동물 모델에 의존하게 되었습니다.
NEC의 동물 모델은 생쥐, 쥐, 새끼 돼지, 토끼 및 개코원숭이에 대해 확립되었다 5,8,9,11. 동물 모델의 강점은 NEC와 유사한 장 질환이 생물 불균형 마이크로바이옴, 저산소증의 반복적인 에피소드 및 모유 수유 부족을 포함하여 인간의 NEC 발병과 관련된 요인에 의해 유발된다는 것입니다 5,8,10,11. 또한, 실험 NEC 동안 관찰 된 염증 반응 및 병리학적 변화는 인간 질병과 유사합니다 5,9,12. 이러한 모델은 인간 NEC의 많은 특징을 모방하지만 동물과 인간에서 NEC의 병태생리학 사이에는 고유한 차이점이 있습니다. 예를 들어, NEC의 쥐 모델은 만삭으로 태어난 마우스에서 유도되며, 장 발달이 불완전하지만 NEC의 병태생리학은 이러한 임상적 맥락에서 본질적으로 다릅니다. 출생 시 쥐의 장 유전자 발현은 생존 가능한 인간 태아와 유사하며, 임신 22-24주의 조산아의 유전자 발현과 14일째까지 비슷하지 않다(P14)13. 이것은 P10 이후 마우스에서 장 손상이 일반적으로 유도될 수 없기 때문에 쥐 NEC 모델을 혼란스럽게 합니다. 또한, 생쥐의 근친교배 균주는 인간 신생아15의 면역학적 다양성14 및 미생물학적 다양성이 결여되어 있으며, 이는 또 다른 교란 요인으로 작용한다. 따라서 NEC 연구에 1차 인간 샘플의 통합이 증가하면 이 분야 연구의 임상적 관련성이 향상됩니다.
NEC의 체외 기전에 대한 연구는 전통적으로 대장 선암(Caco2) 및 인간 결장 선암(HT-29) 세포와 같은 성인 장암 세포에서 유래한 단일형 세포주를 활용해 왔다16. 이러한 모델은 편리하지만 성인 암 세포로부터의 성장, 분극되지 않은 구조 및 배양의 반복적인 통과와 관련된 표현형 변화로 인해 생리학적 관련성이 제한적입니다. 장 장내골은 장 조직의 소낭에서 성장할 수 있고, 모든 장 상피 아형으로 분화될 수 있으며, 3차원(3D) 융모와 같은 구조를 형성할 수 있기 때문에 이러한 모델보다 개선된다(17,18,19,20). 최근에, 장내 장내골은 미세유체 기술과 결합되어 소장-온-어-칩(small intestine-on-a-chip) 모델을 개발하고, 생리학적으로 더 관련성이 높은 시험관 내 모델 시스템(in vitro modelsystem)을 제공한다(21).
초기 장기 칩 미세 유체 장치는 2000 년대 초반에 도입되었습니다22,23,24. 최초의 장기 칩(organ-on-a-chip) 모델은 인간 호흡 폐 온 칩 25(Lung-on-a-chip25)였습니다. 그 뒤를 이어 장 21, 간26, 신장27, 골수 28, 혈액뇌장벽29, 심장30과 같은 수많은 단일 장기 모델이 나왔다. 이러한 장기 칩 모델은 급성 방사선 증후군,31 만성 폐쇄성 폐 질환,32 및 신경 퇴행성 질환33을 포함한 급성, 만성 및 희귀 질환을 연구하는 데 사용되었습니다. 이러한 칩에 있는 세포의 분극화된 특성과 다공성 막으로 분리된 두 개의 세포 구획의 존재는 관류, 화학 농도 구배 및 면역 세포 화학주성34,35와 같은 복잡한 생리학적 과정의 모델링을 가능하게 합니다. 따라서 이러한 미세유체 시스템은 인간 질병의 병태생리학 및 메커니즘을 연구하기 위한 새로운 도구를 제공합니다.
소장온어칩(small intestine-on-a-chip) 모델은 2018년 Kasendra et al.에 의해 기술되었으며, 그는 소아(10-14세) 소장 생검 표본을 장내형으로 분화하고 미세유체 장치(21)에서 배양했습니다. 혈관 내피 세포, 지속적인 배지 흐름 및 스트레칭/이완도 이 모델에 통합되었습니다. 그들은 장 상피 아형 분화, 3D 융모 유사 축의 형성, 점액 생성 및 소장 유전자 발현 패턴을 관찰했다21. 이 미세유체 모델은 NEC36을 가진 신생아 장 장내, 내피 세포 및 신생아의 마이크로바이옴을 통합하는 NEC-on-a-chip 시스템의 개발로 신생아 질환에 적용되었습니다. NEC-on-a-chip은 염증성 유전자 발현, 특수 상피 세포의 손실, 장 장벽 기능 감소 등 인간 NEC의 많은 중요한 특징을 요약합니다36. 따라서 이 모델은 기계론적 연구 및 약물 발견을 포함하여 NEC 연구에서 수많은 응용 분야를 가지고 있습니다. 이 원고에서는 NEC-on-a-chip 모델의 성능에 대한 자세한 프로토콜을 제공합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Leu15]-Gastrin I human</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td></td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium/Ham&#39;s F-12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free (50x)</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Bio-kit</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy 2 Multi-Mode Microplate Reader</td>
			<td>Agilent</td>
			<td>&#160;7131000</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND Methacrylate (PMMA) Cuvettes, Semi-Micro</td>
			<td>BrandTech</td>
			<td>759085D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354270</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Opus Real-Time PCR Systems</td>
			<td>Bio-Rad</td>
			<td>12011319</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip Cradle</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip-S1 Stretchable Chip</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Sigma-Aldrich</td>
			<td>SML1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear TC-treated Multiple Well Plates,&#160; 48 well&#160;</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from human placenta</td>
			<td>Sigma-Aldrich</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type I, powder</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Human Endothelial Cell Medium with Kit&#160;</td>
			<td>Cell Biologics</td>
			<td>H-1168</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Polypropylene Centrifuge Tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Polypropylene Centrifuge Tubes, 50mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen&#160;</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II automated cell counter</td>
			<td>Invitrogen&#160;</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma-Aldrich</td>
			<td>D5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Cascade Blue, 3000 MW, Anionic, Lysine Fixable</td>
			<td>Invitrogen</td>
			<td>&#160;D7132</td>
			<td>Permeability dye&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PES Filter Units, 0.2um aPES membrane</td>
			<td>Fisher Scientific</td>
			<td>FB12566504</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Modified Eagle&#8242;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5 M,&#160; pH 8.0</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-1 surface activation reagent</td>
			<td>Emulate</td>
			<td>ER-1</td>
			<td>Chip Activation Reagent 1</td>
		</tr>
		<tr>
			<td>ER-2 surface activation reagent&#160;</td>
			<td>Emulate</td>
			<td>ER-2</td>
			<td>Chip Activation Reagent 2</td>
		</tr>
		<tr>
			<td>Fibronectin Human Protein, Plasma</td>
			<td>Gibco</td>
			<td>33016015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Petri Dishes with Clear Lid, 100mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin-Based Coating Solution&#160;</td>
			<td>Cell Biologics</td>
			<td>6950</td>
			<td></td>
		</tr>
		<tr>
			<td>Genie Temp-Shaker 300</td>
			<td>Scientific Industries, Inc.</td>
			<td>SI-G300</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin&#160;</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, Liquid 1M Solution (238.3 mg/ mL)</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate&#160;</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Collagen Type I</td>
			<td>Sigma-Aldrich</td>
			<td>CC050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Primary Small Intestinal Microvascular Endothelial Cells</td>
			<td>Cell Biologics</td>
			<td>H-6054</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Fisher Scientific</td>
			<td>03-000-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp General Purpose Deluxe Water Baths</td>
			<td>Fisher Scientific</td>
			<td>FSGPD10</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth (LB) agar, Miller</td>
			<td>Supelco</td>
			<td>L3027</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN Cells&#160;</td>
			<td>American Type Culture Collection</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Basement Membrane Matrix, LDEV-free&#160;</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Cell Culture Matrix</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>1009005</td>
			<td></td>
		</tr>
		<tr>
			<td>NAILSTAR UV LAMP</td>
			<td>NailStar</td>
			<td>NS-01-US</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop OneC&#160;Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-274200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>72340</td>
			<td></td>
		</tr>
		<tr>
			<td>Orb-HM1 Hub Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>ThermoFisher</td>
			<td>047392.9L</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L8-200XLS+</td>
			<td>Rainin</td>
			<td>17013805</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips TR LTS 1000&#181;L S 768A/8</td>
			<td>Rainin</td>
			<td>17014966</td>
			<td></td>
		</tr>
		<tr>
			<td>Pod Portable Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Grade Fetal Bovine Serum (FBS)(Heat Inactivated)&#160;</td>
			<td>Avantor Seradigm</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Reverse Transcription Kit&#160;</td>
			<td>QIAGEN</td>
			<td>205313</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>SB 431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>Square BioAssay Dish with Handles, not TC-treated&#160;</td>
			<td>Corning</td>
			<td>431111</td>
			<td></td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR&#160;Green Supermix</td>
			<td>Bio-Rad</td>
			<td>1725271</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GV Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainers, 70um</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringes, 10mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-453</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight, fine, sharp point scissors</td>
			<td>Miltex&#160;Instruments</td>
			<td>MH5-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Sorvall X4R Pro-MD Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75016052</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>TRIzol Reagent&#160;</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>RNA extraction reagent</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4% (w/v) in PBS, pH 7.5 &#177; 0.5</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), no phenol red&#160;</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>Enzymatic Dissociation Reagent</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>VIOS 160i CO2 Incubator, 165 L</td>
			<td>Thermo Scientific</td>
			<td>13-998-252</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Zo&#235;-CM1 Culture Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic model of necrotizing enterocolitis incorporating human neonatal intestinal enteroids and a dysbiotic microbiome

괴사성 장염(Necrotizing enterocolitis, NEC)은 심각하고 치명적일 수 있는 장 질환으로, 복잡한 발병 기전으로 인해 연구가 어려웠으며, 아직 완전히 이해되지 않고 있습니다. NEC의 병태생리학에는 장 밀착 접합부의 파괴, 장 장벽 투과성 증가, 상피 세포 사멸, 미생물 미생물 미생물 불균형 및 조절 장애 염증이 포함됩니다. NEC를 연구하는 전통적인 도구에는 동물 모델, 세포주, 인간 또는 생쥐의 장 오가노이드가 포함됩니다. 이러한 모델 시스템을 사용한 연구는 질병 병태생리학에 대한 현장의 이해를 향상시켰지만, 인간 NEC의 복잡성을 요약하는 능력은 제한적입니다. NEC-on-a-chip이라고 하는 미세유체 기술을 사용하여 NEC의 개선된 시험관 모델이 현재 개발되었습니다. NEC-on-a-chip 모델은 인간 내피 세포 및 중증 NEC를 앓고 있는 유아의 마이크로바이옴과 함께 배양된 조산아에서 유래한 장내 장내형 장내형 물질로 파종된 미세유체 장치로 구성됩니다. 이 모델은 NEC의 병태생리학에 대한 기계론적 연구를 위한 귀중한 도구이자 신생아 장 질환에 대한 약물 발견 테스트를 위한 새로운 리소스입니다. 이 원고에서는 NEC-on-a-chip 모델에 대한 자세한 설명이 제공됩니다.

Necrotizing enterocolitis (NEC) affects preterm infants, with an incidence of up to 10% in those born weighing &#60; 1500 g1. The pathophysiology of NEC is complex and includes damage to the intestinal epithelium, disruption of intestinal tight junctions, increased gut barrier permeability, immune dysregulation, and epithelial cell death2,3. Our understanding of the mechanisms involved in the pathogenesis of NEC remains incomplete, and despite decades of research, there are still no effective targeted therapies.
A significant barrier to advancing NEC research is the limited availability and small size of primary intestinal tissue isolated from human infants. Intestinal tissue resected from infants with NEC is often necrotic and severely damaged, which complicates studies into mechanisms that precede disease onset. For example, the small intestine of infants with NEC is inundated with immune cells, and a reduced number of intestinal stem cells, decreased epithelial cell proliferation, and increased epithelial cell apoptosis are also observed4,5,6,7. This leads to difficulties in culturing intestinal epithelial cells from these samples and in isolating RNA and proteins, which can be degraded in this hostile inflammatory environment. Additionally, since the disease process is already advanced in infants with surgical NEC, mechanistic studies into factors that induce disease are unfeasible. These limitations have led to a reliance on animal models for mechanistic studies of NEC.
Animal models of NEC have been established for mice, rats, piglets, rabbits, and baboons5,8,9,11. A strength of animal&#160;models is that NEC-like intestinal disease is induced by factors associated with NEC onset in humans, including a dysbiotic microbiome, repeated episodes of hypoxia, and the absence of breast milk feeds5,8,10,11. In addition, the inflammatory response and pathologic changes observed during experimental NEC parallel human disease5,9,12. While these models mimic many of the features of human NEC, there are inherent differences between the pathophysiology of NEC in animals and humans. For example, the murine model of NEC is induced in mice born full-term, and although their intestinal development is incomplete, the pathophysiology of NEC is inherently different in this clinical context. Murine intestinal gene expression at birth is similar to a pre-viable human fetus and does not approximate that of a preterm neonate of 22-24 weeks gestation until day 14 (P14)13. This confounds the murine NEC model because intestinal injury cannot generally be induced in mice after P10. In addition, inbred strains of mice lack the immunologic14 and microbiologic diversity of human neonates15, which serves as another confounding factor. Thus, increased incorporation of primary human samples into NEC research improves the clinical relevance of studies in this field.
Studies into the mechanisms of NEC in vitro have traditionally utilized monotypic cell lines derived from adult intestinal cancer cells, such as colorectal adenocarcinoma (Caco2) and human colon adenocarcinoma (HT-29) cells16. These models are convenient but limited in physiologic relevance due to their growth from adult cancer cells, non-polarized architecture, and phenotypic changes related to repeated passages in culture. Intestinal enteroids improve upon those models since they can be grown from the crypts of intestinal tissue, differentiated into all intestinal epithelial subtypes, and form a three-dimensional (3D) villus-like structure17,18,19,20. Recently, intestinal enteroids have been combined with microfluidic technology to develop a small intestine-on-a-chip model and provide a more physiologically relevant in vitro model system21.
The initial organ-on-a-chip microfluidic devices were introduced in the early 2000s22,23,24. The first organ-on-a-chip model was the human breathing lung-on-a-chip25. This was followed by numerous single-organ models such as intestine21, liver26, kidneys27, bone marrow28, blood-brain barrier29, and heart30. These organ-on-a-chip models have been used to study acute, chronic, and rare diseases, including acute radiation syndrome,31 chronic obstructive pulmonary disease,32 and neurodegenerative diseases33. The polarized nature of the cells on these chips and the presence of two cellular compartments separated by a porous membrane allows for the modeling of complex physiologic processes such as perfusion, chemical concentration gradients, and immune cell chemotaxis34,35. These microfluidic systems thus provide a new tool for studying the pathophysiology and mechanisms of human disease.
The small intestine-on-a-chip model was described by Kasendra et al. in 2018, who utilized pediatric (ages 10-14 years old) small intestinal biopsy specimens differentiated into enteroids and cultured on a microfluidic device21. Vascular endothelial cells, continuous media flow, and stretch/relaxation were also incorporated into this model. They observed intestinal epithelial subtype differentiation, formation of 3D villus-like axes, mucus production, and small intestinal gene expression patterns21. This microfluidic model was applied to neonatal disease with the development of the NEC-on-a-chip system, which incorporates neonatal intestinal enteroids, endothelial cells, and the microbiome from a neonate with NEC36. NEC-on-a-chip recapitulates many of the critical features of human NEC, including inflammatory gene expression, loss of specialized epithelial cells, and reduced gut barrier function36. Thus, this model has numerous applications in the study of NEC, including mechanistic studies and drug discovery. In this manuscript, a detailed protocol for the performance of the NEC-on-a-chip model is provided.

저자 스포트라이트: NEC-on-a-Chip 모델을 통한 이해 및 치료 전략 강화 

인간 신생아 장 장내염과 디스바이오틱 마이크로바이옴을 통합한 괴사성 장염의 미세유체 모델

Research in this field is challenging because the pathophysiology of necrotizing enterocolitis is complex, and this complexity is not reflected in many commonly used in vitro models. Studies using human samples are often limited by small sample volumes and the rarity of samples obtained at the time of intestinal surgery. Our goal is to determine the mechanisms that lead to the development of NEC in preterm infants and to identify new treatment strategies.The NEC-on-a-Chip system improves upon previous models by more closely reflecting the physiology of the neonatal intestine. In this model, we combine patient-derived intestinal organoids, endothelial cells, and the microbiome from a neonate who had severe NEC onto the microfluidic chip.

엔테로이드를 미세유체 장치(그림 1)에 파종하고 상술한 바와 같이 배양하였다. 파종 전 세포 배양 매트릭스 하이드로겔에서 엔테로이드의 성장과 파종 후 장 상피 세포 단층의 후속 확장은 명시야 현미경을 통해 모니터링되었습니다(그림 2). 합류하는 장 상피 세포 단층이 형성된 후 성숙한 3D 융모와 같은 구조로 발전했습니다(그림 2</s...

Watch this Scientific Journal Video about Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model at JoVE.com

이 프로토콜은 질병 발병에 대한 기계론적 연구에 사용할 수 있는 괴사성 장염(NEC)의 시험관 내 모델을 설명합니다. 인간 신생아 장, 내피 세포 및 중증 NEC가 있는 신생아의 장내 마이크로바이옴에서 추출한 장내 장내형 칩을 파종한 것이 특징입니다.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

괴사성 장염의 병태생리학이 복잡하고 이러한 복잡성이 일반적으로 사용되는 많은 체외 모델에 반영되지 않기 때문에 이 분야의 연구는 어렵습니다. 인간 검체를 사용한 연구는 소량의 검체량과 장 수술 시 채취한 검체의 희소성으로 인해 제한되는 경우가 많습니다. 우리의 목표는 조산아에서 NEC의 발달을 유도하는 메커니즘을 결정하고 새로운 치료 전략을 식별하는 것입니다.NEC-on-a-Chip 시스템은 신생아 장의 생리학을 보다 밀접하게 반영하여 이전 모델을 개선합니다. 이 모델에서는 환자 유래 장 오가노이드, 내피 세포 및 중증 NEC를 가진 신생아의 마이크로바이옴을 미세유체 칩에 결합합니다.

인간 신생아 장 장내염과 디스바이오틱 마이크로바이옴을 통합한 괴사성 장염의 미세유체 모델

Abstract