通过水射流技术注射猪脂肪组织衍生的基质细胞
Summary
我们提出了一种通过无针水射流技术进行细胞注射的方法，以及细胞活力，增殖和弹性测量方面的交付后研究的后遗症。
Abstract
尿失禁（UI）是一种非常普遍的疾病，其特征在于尿道括约肌缺乏。再生医学分支，特别是细胞疗法，是改善和恢复尿道括约肌功能的新方法。尽管在临床环境中常规通过针头和注射器注射活性功能细胞，但这些方法具有显着的缺点和局限性。在这种情况下，无针水刀（WJ）技术是一种可行且创新的方法，可以通过视觉引导膀胱镜在尿道括约肌中注射活细胞。在本研究中，我们使用WJ将猪脂肪组织来源的基质细胞（pADSC）递送到尸体尿道组织中，并随后研究了WJ递送对细胞产量和活力的影响。我们还通过原子力显微镜（AFM）测量评估了生物力学特征（即弹性）。我们发现，WJ递送的pADSC的细胞弹性显着降低。与对照组相比，存活率明显较低，但仍高于80%。
Introduction
尿失禁 （UI） 是一种广泛存在的疾病，在欧洲人群中患病率为 1.8 – 30.5%，主要表现为尿道括约肌功能障碍。从临床角度来看，当保守疗法或物理治疗无法解决和缓解新出现的症状时，通常会向患者提供手术治疗。
用于括约肌复合物故障的潜在再生修复的细胞疗法已成为治疗UI病理学的前卫方法2，3。其主要目标是替换，修复和恢复受损组织的生物功能。在UI的动物模型中，干细胞移植在尿动力学结果中显示出有希望的结果2，4，5。干细胞作为最佳细胞候选者出现，因为它们具有经历自我更新和多能分化的能力，从而有助于受影响的组织再生6。尽管即将到来的再生潜力，但细胞疗法的实际使用仍然受到阻碍，因为细胞的微创递送仍然面临有关注射精度和靶标覆盖率的几个挑战。尽管目前用于细胞递送的方法是通过针刺系统7注射，但它通常会导致活细胞的整体缺陷，据报道移植后的活力低至1%-31%8。此外，通过针头注射的细胞递送也被证明会影响放置，保留率，以及移植细胞向靶组织9，10，11的分布。克服上述限制的一种可行的新颖方法是通过水射流技术进行无针细胞递送。
水射流（WJ）技术正在成为一种新方法，它能够在尿道括约肌12，13的视觉控制下通过膀胱镜高通量递送细胞。WJ可在E5至E8013的不同压力（E = 以巴为单位的效应）下进行细胞递送。在第一阶段，（组织渗透阶段）用高压（即E60或E80）施加等渗溶液，以松动靶向组织周围的细胞外基质并打开小的互连微腔。在第二阶段（注射阶段），压力在几毫秒内降低（即，高达E10），以便将细胞轻轻地输送到靶组织中。在这种两步相施用之后，细胞在射出时不会受到针对组织的额外压力，而是以低压流漂浮到充满液体的海绵状区域13中。在通过WJ将干细胞注射到尸体尿道组织中的离体模型设置中，活细胞随后可以从组织中抽吸并取出并在体外进一步扩增13。尽管Weber等人2020年的一项研究证明了WJ将无足迹心肌细胞输送到心肌14中的可行性和适用性，但必须记住，WJ技术仍处于原型阶段。
以下方案描述了如何制备和标记猪脂肪组织来源的基质细胞（pADSC），以及如何通过WJ技术和威廉姆斯膀胱镜检查针（WN）将它们递送到捕获液和尸体组织中。细胞注射后，通过原子力显微镜（AFM）评估细胞活力和弹性。通过分步说明，该协议提供了一种清晰简洁的方法来获取可靠的数据。讨论部分介绍并描述了该技术的主要优点，局限性和未来前景。WJ细胞递送以及这里报道的翻译后遗症分析正在取代标准的针头注射，并为靶组织的再生愈合提供固体细胞递送框架。在我们最近的研究中，我们提供的证据显示，与针头注射相比，WJ更精确地递送细胞，并且至少具有相当的活力15，16。
Protocol
猪脂肪组织样本是从图宾根大学实验外科研究所获得的。所有程序均由当地动物福利机构批准，动物实验编号为CU1/16。 1. 猪脂肪组织源性基质细胞的分离 使用从实验外科研究所在50 mL离心管中输送到实验室的猪脂肪组织。 将组织转移到无菌工作台下的无菌培养皿中，并用两把手术刀（10号）将其切碎成小碎片和糊状物。注意：也可以使用剪刀来…
Representative Results
通过两种方法递送细胞后，与使用E60-10设置的WJ注射相比，通过WN递送的细胞的活力（97.2 ±±2%，n = 10，p<0.002）更高（图2）。生物力学评估结果表明：捕获液中细胞的WN注射与对照组（1.176 kPa）相比，弹性模量（EM;0.992 kPa）无显着差异; 图3A），而WJ注射触发了细胞EM的显着降低（0.440 kPa，p<0.001， 图3B）。注意到WJ注射后EM降低40-50%?…
Discussion
在本研究中，我们展示并提出了WJ细胞递送程序的分步方法，并采用定量研究的后遗症来评估WJ递送对细胞特征的影响：细胞活力和生物力学特征（即EM）。注射WJ后，85.9%的收获细胞是活的。在WN注射方面，97.2%的细胞在注射后保持其活力。因此，WJ方法满足了临床实施的绝对要求：超过80%的活细胞在递送后18。虽然使用WJ方法实现了标准化和可重复的方案，但针头注射的结果高度?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢原始出版物的合著者的帮助和支持。
Materials
| 50 mL centrifuge tube | Greiner BioOne | 227261 | |
| 1 mL BD Luer-LokTM Syringe | BD Plastik Inc | n.a. | |
| 100 µm cell sieve | Greiner BioOne | 542000 | |
| 15 mL centrifuge tube | Greiner BioOne | 188271 | |
| 75 cm2 tissue culture flask | Corning Incorporated | 353136 | |
| AFM head | (CellHesion 200) JPK | JPK00518 | |
| AFM processing software | Bruker | JPK00518 | |
| AFM software | Bruker | JPK00518 | |
| AFM system Cell Hesion 200 | Bruker | JPK00518 | |
| All-In-One-Al cantilever | Budget Sensors | AIO-10 | tip A, Conatct Mode, Shape: Beam Force Constant: 0.2 N/m (0.04 – 0.7 N/m) Resonance Frequency: 15 kHz (10 – 20 kHz) Length: 500 µm (490 – 510 µm) Width: 30 µm (35 – 45 µm) Thickness: 2.7 µm (1.7 – 3.7 µm) |
| Amphotericin B solution | Sigma | A2942 | 250 µg/ml |
| Atomic Force Microscope (AFM) | CellHesion 200, JPK Instruments, Berlin, Germany | JPK00518 | |
| BD Microlance 3 18G | BD | 304622 | |
| bovine serum albumin | Gibco | A10008-01 | |
| Cantilever | All-In-One-AleTl, Budget Sensors, Sofia, Bulgaria | AIO-TL-10 | tip A, k ¼ 0.2 N/m |
| C-chip disposable hemocytometer | NanoEnTek | 631-1098 | |
| centrifuge: Rotina 420R | Hettich Zentrifugen | ||
| Collagenase, Type I, powder | Gibco | 17100-017 | |
| Dulbecco’s Modified Eagle’s Medium – low glucose | Sigma | D5546 | |
| Feather disposable scalpel (No. 10) | Feather | 02.001.30.010 | |
| fetal bovine serum (FBS) | Sigma | F7524 | |
| HEPES sodium salt solution (1 M) | Sigma | H3662 | |
| Inverted phase contrast microscope (Integrated with AFM) | AxioObserver D1, Carl Zeiss Microscopy, Jena, Germany | L201306_03 | |
| laboratory bags | Brand | 759705 | |
| Leibovitz's L-15 medium without l-glutamine | Merck | F1315 | |
| Leibovitz's L-15 medium without L-glutamine | (Merck KGaA, Darmstadt, Germany) | F1315 | |
| L-glutamine | Lonza | BE 17-605C1 | 200 mM |
| LIVE/DEADTM Viability/Cytotoxicity Kit | Invitrogen by Thermo Fisher Scientific | L3224 | Calcein AM and EthD-1 are used from this kit. |
| Microscope software: Zen 2.6 | Zeiss | ||
| Microscope: AxioVertA.1 | Zeiss | ||
| Nelaton-Catheter female | Bicakcilar | 19512051 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | 10000 U/ml Penicillin 10000 µg/ml Streptomycin |
| Petri dish heater associated with AFM | Bruker | T-05-0117 | |
| Petri dish heater associated with AFM | JPK Instruments AG, Berlin, Germany | T-05-0117 | |
| Phosphate buffered saline (PBS) | Gibco | 10010-015 | |
| Statistical Software: SPSS Statistics 22 | IBM | ||
| Sterile Petri dish – CellStar | Greiner BioOne | 664160 | |
| Tissue culture dishes | TPP AG | TPP93040 | |
| Tissue culture dishes | TPP Techno Plastic Products AG, Trasadingen, Switzerland | TPP93040 | |
| Trypan Blue 0.4% 0.85% NaCl |
Lonza | 17-942E | |
| Trypsin-EDTA solution | Sigma | T3924 | |
| Waterjet: ERBEJET2 device | Erbe Elektromedizin GmbH | ||
| Williams Cystoscopic Injection Needle | Cook Medical | G14220 | 23G, 5.0 Fr, 35 cm |
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Cite This Article
Knoll, J., Danalache, M., Linzenbold, W., Enderle, M., Abruzzese, T., Stenzl, A., Aicher, W. K. Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology. J. Vis. Exp. (177), e63132, doi:10.3791/63132 (2021).