Summary

生产和治疗间充质干行政/基质细胞(MSC)球体底漆在三维培养下无异物条件

Published: March 18, 2017
doi:

Summary

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or ‘spheroids’ of MSCs primed under xeno-free conditions for experimental and clinical applications.

Abstract

间充质干/基质细胞(MSCs)持有生物工程和再生医学很大的希望。的MSC可以从多个成人组织通过其强粘附到组织培养塑料中分离,然后在体外进一步扩大,最常使用的胎牛血清(FBS)。因为FBS可引起干细胞成为免疫原性的,其中的MSC培养物的存在限制了细胞的临床和实验的应用程序。因此,研究用人化学定义无异物(XF)媒体MSC文化是非常有价值的。 MSC的许多有益效果已被归因于它们对调节炎症和免疫能力,主要通过免疫调节因子的分泌,如肿瘤坏死因子刺激的基因6(TSG6)和前列腺素E2(PGE 2)。然而,干细胞需要活化以产生这些因素,并且自MSC的效果往往短暂的,极大的兴趣已经出现以发现的激活预细胞PRIO方式R以它们的使用,从而消除了滞后时间用于体内活化。这里,我们提出的协议能够有效地激活或首要的MSC在三维(3D)培养物 化学上确定的XF的条件下,并施用在体内这些预活化的MSC。具体地讲,我们首先描述的方法生成使用XF介质球形MSC微组织或在悬滴“球体”和演示如何球体和条件培养基(CM)可收获用于各种应用。第二,我们描述的基因表达的屏幕和在体外功能测定迅速评估MSC活化在球状体的水平,强调细胞的抗炎和抗肿瘤潜力。第三,我们描述了一种新的方法来注入完好的MSC球体成用于体内效力测试小鼠腹腔。总体来说,此方案得到克服根据化学定义XF CON预激活干细胞的主要挑战条件和提供了一个灵活的系统管理MSC球体的疗法。

Introduction

间充质干细胞/基质细胞(MSCs)已经显示关于各种再生医学的方法的巨大潜力。的MSC最初分离的骨髓的基质组分,但至今已从许多其它成体组织,包括脂肪组织1,2,3获得。有趣的是,主要分离方法涵盖MSCs的显着属性,以紧密附着到组织培养塑料中的胎牛血清(FBS)的存在。虽然这种传统的隔离技术允许在二维(2D)培养容易和迅速扩展的MSC,它也很人工和无视导致重要的细胞特征4潜在损失的天然三维(3D)环境的意义,5 6。因此,干细胞在三维文化的研究,比传统的二维培养更符合生理,已经出现在搜索“丢失/减弱”MSC特性。此外,极大的兴趣已经上升到识别(XF)无异物化学定义的条件MSC文化和活化,从而使细胞临床应用更适合。

许多研究已经发表表明无论在生物材料和球形聚集体或球状体MSC的3D培养。在生物材料的MSC最初设计用于组织工程的方法与细胞种子支架替换损坏的组织,而MSC的球体培养物被视为一种方式来理解用于在临床前或临床试验中的治疗的细胞给药后在体内的MSC行为4,5,7。有趣的是,干细胞形式球状体自发时不允许粘附到组织培养塑料8,9,10。传统上,细胞聚集是由旋转烧瓶方法或液体覆盖技术,最初用于癌症生物学中的努力,试图模仿肿瘤微环境的方法促进。最近,其他方法已经浮现演示在培养皿的细胞聚集预涂具有特定的化学物质,以防止细胞-塑料粘附4,5,6。之一,以产生MSC球状体的最简单和最经济的方法是将培养他们在悬滴,这是经常用于产生从胚胎干细胞的胚状体的技术。与悬滴培养技术,细胞粘附于组织培养塑料是通过在介质的上的组织培养皿盖的下侧下降的细胞悬浮并允许重力促进细胞aggreg防止通货膨胀在下拉的顶点。球体大小可以通过改变细胞浓度或者滴体积,使得悬滴培养尤其容易控制可以容易地操纵。

关于MSC的3D培养早期的研究表明在细胞中的3D相比,他们的2D对应6,8,9的特性基团的差异。同时,报告表明,干细胞在体内的有益效果依靠其成为通过微环境线索激活,并且作为响应,以产生抗炎和免疫调节因子11的能力。有趣的是,许多这些因素,如前列腺素E2(PGE 2),肿瘤坏死因子刺激的基因6(TSG6)和肝细胞生长因子(HGF)是在更大的数量由MSC球体比传统的二维的MSCs铺平了道路产生的理念使用3D培养激活细胞8,12,13。此外,基因活化在3D培养出现注射入小鼠12后概括机制,至少部分,细胞活化。通过激活之前将其在实验中使用的MSC,该细胞的作用可以被延长,更突出在体内传统的MSC效果往往延迟和短暂的,并且可以被描述为“打并运行”。在过去的几年中,使用MSC球状体重要的功能性研究已经表明,它们可以抑制炎症反应和通过影响效应细胞如巨噬细胞,树突细胞,嗜中性粒细胞和T细胞使得球状体致敏的MSC 2的一个有吸引力的形式调节体内免疫,3。此外,生产抗癌分子,如int的erleukin-24(IL-24),肿瘤坏死因子相关凋亡诱导配体(TRAIL),在干细胞的相对的三维培养物提高到单层的MSC,这可能对靶向癌症疗法8,10,14被利用的现象。

由于传统的MSC文化不仅需要利用组织培养塑料也FBS,另一个障碍,使MSC球体更适合临床使用已被克服。为了解决这一难题,我们最近发现形成的MSC球状体的特定化学成分确定的XF的条件下,建立所得到的MSC球体被激活以产生相同的抗炎和抗肿瘤分子作为在条件与FBS 14产生的球状体。这里,这些研究结果在演示中使用XF 3D培养预活化的MSCs的产生几个详细方案提出媒体。此外,协议呈现描述评估MSC的活化水平的问候其抗炎,免疫调节和抗肿瘤作用,与递送完整球体到小鼠的实用方法一起有效的方法。

Protocol

1. MSC分离和扩增获得早期中心代MSCs编制和成体干细胞(分布http://medicine.tamhsc.edu/irm/msc-distribution.html )15作为冷冻瓶。另外,隔离骨髓穿刺以下例行协议14 MSC和存储为冷冻小瓶。 制备完全培养基(CCM),它是最小必需培养基α(αMEM)补充有15-20%的溢价选择FBS,2mM L-谷氨酰胺和1x青霉?…

Representative Results

在目前的工作中,采用悬滴文化生成球形紧凑微型组织或XF条件下激活干细胞的“球体”。 图1中的试验用路线图描绘的MSCs被鼓励自组装悬浮于悬滴72小时,在此之后,球状体,或在CM装入球体源性治疗因子时变成球体,可以收集和潜在地在两个利用研究和临床应用。大量需要的球生产细胞可以在一个星期内通过以低密度接种的干细胞,通常100-200细胞每厘米<sup…

Discussion

在一些研究和临床应用中使用的最佳的MSC应高度活化以最大化他们的利益,和化学成分确定的XF的条件下优先制备,以尽量减少潜在抗原从异种介质组分如胎牛血清的交付。在这里所描述的协议中,我们已经表明方法1)通过形成球状体的激活在三维培养的MSC,2)实现的MSCs XF条件下的3D激活,3)评价在关于他们抗球体MSCs的激活水平抗炎,免疫调节和抗肿瘤潜力,和4)提供激活的MSC作为完整的球体…

Divulgations

The authors have nothing to disclose.

Acknowledgements

This work was funded in part by grant P40RR17447 from the National Institute of Health and award RP150637 from the Cancer Prevention and Research Institute of Texas. We would like to thank Dr. Darwin J. Prockop for his support on the project.

Materials

MEM-α (minimal essential medium alpha) ThermoFisher/Gibco 12561049; 12561056; 12561072 minimal essential medium for preparation of MSC growth medium (CCM)
FBS (fetal bovine serum), premium select Atlanta Biologicals S11595; S11510; S11550; S11595-24 component of complete culture media for all types of cells
L-glutamine ThermoFisher/Gibco 25030081; 25030149; 25030164 component of complete culture media for all types of cells
Penicillin/Streptomycin ThermoFisher/Gibco 15070063 component of complete culture media for all types of cells
Sterilization Filter Units, 0.22 µm PES membrane MilliporeSigma SCGPU01RE; SCGPU02RE; SCGPU05RE; SCGPU10RE; SCGPU11RE media sterilization
150 mm cell culture dish Nunc D8554 SIGMA cell culture
Thermo Forma water-jacketed CO2 humidified incubator Thermo Fisher Model 3110 incubation of cultured cells
Early passage MCSs Center for the preparation and Distribution of Adult Stem Cells at The Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White NA preparation of 2D and 3D cultures of MSCs
water bath VWR 89501-468 warming media to 37 °C
Pipettes Eppendorf 492000904 manual liquid handling
Pipete-Aid Drummond Scientific Company 4-000-300 handling sereological pipetes
Costar sterile serological pipet (5, 10, 25 and 50 ml) Corning 4487; 4101; 4251; 4490 liquid handling
PBS (phosphate buffered saline), pH 7.4 ThermoFisher/Gibco 10010023; 10010072; 10010031; 10010049 cell culture processing
0.25% trypsin/EDTA solution ThermoFisher/Gibco 25200056; 25200072; 25200114 lifting adherent cells and dispersing cell aggregates
15 ml conical tube Corning/BD Falcon 352097 cell centrifugation
50 ml conical tube Corning/BD Falcon 352098 cell centrifugation
Eppendor refrigerated centrifuge Eppendorf/Fisher Scientific Model 5810R cell centrifugation
hemocytometer Fisher Scientific 26716 cell counting
trypan blue Sigma-Aldrich T8154 SIGMA dead cell exclusion during cell counting in hemacytometer
Defined xenofree MSC medium-1 (XFM-1) ThermoFisher/Gibco A1067501 Xeno-free media specifically formulated for the growth and expansion of human mesenchymal stem cells
Defined xenofree MSC medium-2 (XFM-2) Stem Cell Technologies 5420 Defined, xeno-free medium for human mesenchymal stem cells
HSA (Human serum albumin) Gemini 800-120 Component of xeno-free MSC media
rHSA (recombinant Human serum albumin) Sigma-Aldrich A9731 SIGMA Component of xeno-free MSC media
8-channel pipette, 10 – 100 µL Eppendorf 022453904 preparation of hanging drops
Total RNA isolation Mini Kit Qiagen 74104 Total RNA extraction
Qiashredder Qiagen 79654 Sample homogenization prior to total RNA extraction
RNAse-free DNase Set  Qiagen 79254 On-column DNA elimination during total RNA extraction
β-mercaptoethanol  Sigma-Aldrich M6250 ALDRICH inhibition of RNAses in RLT buffer
Vortex VWR 97043-562 mixing sample
Spectrophotometer Biorad NA RNA concentration and quality
High capacity cDNA Reverse Transcription Kit ThermoFisher/Applied Biosystems 4368814 transcription of total RNA into cDNA
Gene Expression Assays ThermoFisher/Applied Biosystems varies primer/probe combination for real-time PCR
Fast Universal PCR Master Mix ThermoFisher/Applied Biosystems 4352042; 4364103; 4366073; 4367846 master mix for real-time PCR reaction
Real-time PCR system (ABI Prism 7900 HT Sequence Detection System) ABI Prizm NA real-time PCR
1.5 ml centrifuge tube Eppendorf 22364111 cell centrifugation, sample collection and storage
(-80°C) freezer Thermo Fisher Model Thermo Forma 8695 sample storage
PGE2 (Prostaglandin E2) ELISA Kit R&D Systems KGE004B estimation of cytokine concentration in the sample
DMEM (Dulbecco’s modified Eagle medium) ThermoFisher/Gibco 10566-016; 10566-024;10566-032 macrophage culture media
J774 mouse macrophages ATCC TIB-67 mouse macrophage cell line
12-well plate Corning 3513 in-vitro macrophage stimulation
LPS (lipopolysaccharide) Sigma-aldrich L4130 in vitro macrophage stimulation
Mouse TNF-a ELISA kit R&D Systems MTA00B estimation of cytokine concentration in the sample
Mouse IL-10 (interleukin 10) ELISA kit R&D Systems M1000B estimation of cytokine concentration in the sample
RPMI-1640 medium ThermoFisher/Gibco 11875-085 splenocyte culture media
BALB/c mice The Jackson Laboratory 651 in vivo spheroid delivery; splenocyte preparation
Anti-Mouse CD3e Functional Grade Purified eBioscience 145-2C11 In vitro splenocyte stimulation
70 μm strainer  Corning 352350 Splenocyte preparation
Red blood cell lysis solution (1x) Affymetrix eBioscience 00-4333 removal of red blood cells during splenocyte isolation
Mouse IFN-y (interferon gamma) ELISA kit R&D Systems MIF 00 estimation of cytokine concentration in the sample
LNCaP prostate cancer cells  ATCC CRL-1740 study the effect of 3D MSCs on cancer cell lines in vitro
DNA-based cell proliferation assay kit ThermoFisher C7026 cell number measurement based on DNA content
NaCl Sigma-Aldrich S5150 component of lysis reagent
EDTA (ethylenediaminetetraacetic acid) ThermoFisher FERR1021 calcium chelator, component of lysis reagent
Rnase A Qiagen 19101 RNA degradation for measurement of DNA
Filter-based multi-mode microplate reader BMG Technology NA Microplate assays (ELISA, cell quantification, e.t.c.)
HBSS (Hanks balanced salt solution), no calcium, no magnesium, no phenol red ThermoFisher/Gibco 14175079 resupsension of MSC spheroids prior to in vivo injections
Isoflurane MWI Vet Supply 502017 Anesthesia for in vivo injections
Oxygen, compressed gas Praxair NA For use with isoflurane
Thermo Forma BSL-2 cabinet Thermo Fisher Model 1385 Sterile cell culture
Safety I.V. catheter/needle stiletto, 20G, 1 inch Terumo SR*FNP2025 Delivery of shperoids into peritoneal cavity
Sterile micropipette tips Eppendorf varies liquid/cells handling

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Ylostalo, J. H., Bazhanov, N., Mohammadipoor, A., Bartosh, T. J. Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions. J. Vis. Exp. (121), e55126, doi:10.3791/55126 (2017).

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