Isolation of Mouse Pancreatic Endothelial Cells

Summary

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

Abstract

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

Introduction

The pancreas, key to metabolic control and homeostasis, is a highly vascularized organ. The pancreas has both endocrine and exocrine functions, controlling the regulation of blood glucose and digestive enzymes, respectively. These two compartments are linked together by the extensive network of pancreatic blood vessels, facilitating the exchange and transport of oxygen, hormones, and enzymes. Critically, this dense capillary network penetrates the Islet of Langerhans, a cluster of hormone-regulating cells within the pancreas responsible for its endocrine function, consisting of the glucagon-secreting alpha (α) cells, the insulin-secreting beta (β) cells and ....

Protocol

Tissue isolation was performed under the approved study protocol #17010 by the Institutional Animal Care and Use Committee (IACUC) of Beckman Research Institute, City of Hope (Duarte, California, USA). Here, we use Tie-2CreERT2;Rosa26-TdTomato mouse line in C57BL/6 background at 8 - 12 weeks of age. In this line, ECs are labeled with TdTomato when induced with tamoxifen as previously described³⁴. However, this protocol can be adapted for all ages of adult mice with different genotypes and genetic .......

Discussion

In this article, we present a protocol for enrichment and isolation of the pECs. Similar to previous EC isolation protocols from other tissues or organs, this protocol consists of three major processes, namely, physical dissociation, enzymatic digestion, and antibody-based EC enrichment. To address the unique challenges in processing the pancreas, we introduced several key adaptations and critical steps within our protocol: 1) a gentle one-step collagenase digestion with a short incubation time, 2) supplementation of hig.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL eppendorf	USA Scientific	1615-5500
10 cm dish	Genesee Scientific	25-202
25G needles	BD	305145
2X Taq Pro Universal SYBR qPCR Master Mix	Vazyme	Q712-03-AA
5 mL eppendorf	Thermo Fisher	14282300
6-well plate	Greiner Bio-One	07-000-208
70 µm strainer	Fisher	22-363-548
Anti-CD31-biotin	Miltenyi Biotech	REA784
Bovine serum albumin heat shock treated	Fisher	BP1600-100
CaCl2	Fisher	BP510
Centrifuge	Eppendorf
Collagen Type 1, from calf skin	Sigma Aldrich	C9791	Attachment reagent in the protocol
Collagenase Type 1	Worthington Bio	LS004197
Countess Automatic Cell Counter	Thermo Fisher
DAPI	Thermo Fisher	D1306	immunofluorescence
Disposable Safety Scalpels	Myco Instrumentation	6008TR-10
DNAse I	Roche	260913
D-PBS (Ca2+,Mg2+)	Thermo Fisher	14080055
Ethanol	Fisher	BP2818-4
Fetal bovine serum	Fisher	10437028
Incubator			Kept at 37 °C 5% CO2
LS Columns	Miltenyi Biotech	130-042-401
M199	Sigma	M2520-1L
MACS MultiStand with the QuadroMACS Separator	Miltenyi Biotech	130-042-303
Medium 199	Sigma Aldrich	M2520-10X
Microbeads anti-biotin	Miltenyi Biotech	130-090-485
Microscope	Leica		To assess cell morphology
Molecular Grade Water	Corning	46-000-CM
NaCl	Fisher	S271-1
New Brunswick Innova 44/44R Orbital shaker	Eppendorf
PECAM1 (CD31) Antibody	Abcam	ab56299	immunofluorescence
PECAM1 (CD31) Antibody	R&D Systems	AF3628
Phosphate Buffered Saline (10X) (no Ca2+,no Mg2+)	Genesee Scientific	25-507-XB
Primer 36B4 Forward mouse	IDT		AGATTCGGGATATGCTGTTGGC
Primer 36B4 Revese mouse	IDT		TCGGGTCCTAGACCAGTGTTC
Primer Kdr Forward mouse	IDT		TCCAGAATCCTCTTCCATGC
Primer Kdr Reverse mouse	IDT		AAACCTCCTGCAAGCAAATG
Primer Nkx6.1 Reverse mouse	IDT		CACGGCGGACTCTGCATCACTC
Primer Nxk6.1 Forward mouse	IDT		CTCTACTTTAGCCCCAGCG
Primer PECAM1 Forward mouse	IDT		ACGCTGGTGCTCTATGCAAG
Primer PECAM1 Reverse mouse	IDT		TCAGTTGCTGCCCATTCATCA
RNase ZAP	Thermo Fisher	AM9780
RNase-free water	Takara	RR036B
Sterile 12" long forceps	F.S.T	91100-16
Sterile fine forceps	F.S.T	11050-10
Sterile fine scissors	F.S.T	14061-11
Tissue Culture Dishes 2cm	Genesee Scientific	25-260
TRIzol reagent	Fisher	15596018
Trypan Blue	Corning	MT25900CI
Trypsin Inhibitor	Roche	10109886001
Tween-20
VE-Cadherin Antibody	Abcam	ab33168	immunofluorescence
Waterbath