3D生物打印光可调水凝胶研究成纤维细胞活化

摘要

本文介绍了如何 3D 生物打印光可调水凝胶来研究细胞外基质硬化和成纤维细胞活化。

摘要

光可调水凝胶可以响应光照在空间和时间上发生转变。将这些类型的生物材料纳入细胞培养平台并动态触发变化，例如增加微环境硬度，使研究人员能够模拟纤维化疾病进展过程中发生的细胞外基质 （ECM） 变化。本文提出了一种用于3D生物打印能够在明胶支持浴内进行两次连续聚合反应的光电可调水凝胶生物材料的方法。通过调节支持浴的pH值来适应悬浮水凝胶的自由形式可逆嵌入（FRESH）生物打印技术，以促进Michael加成反应。首先，将含有聚乙二醇-α-甲基丙烯酸酯（PEGαMA）的生物墨水与细胞可降解交联剂进行化学计量反应，形成软水凝胶。这些软水凝胶随后暴露在光引发剂和光下，以诱导未反应基团的均聚化并使水凝胶变硬。该协议涵盖水凝胶合成、3D 生物打印、光强化和终点表征，以评估 3D 结构内的成纤维细胞活化。这里介绍的方法使研究人员能够3D生物打印各种经历pH催化聚合反应的材料，并可用于设计组织稳态，疾病和修复的各种模型。

引言

3D生物打印是一项变革性技术，使研究人员能够精确地将细胞和生物材料沉积在3D体积内，并重建生物组织的复杂分层结构。在过去的十年中，3D生物打印的进步已经创造了跳动的人类心脏组织¹，肾脏组织的功能模型²，肺内气体交换模型³，以及用于癌症研究的肿瘤模型⁴。嵌入式 3D 生物打印技术的发明，例如悬浮水凝胶的自由形式可逆嵌入 （FRESH） 生物打印，使得在 3D 中再现复杂的软组织结构（如肺血管⁵ 甚至人类心脏⁶ ）成为可能。 FRESH 3D 生物打印有助于通过挤出到剪切稀化支撑槽中逐层打印柔软和低粘度的生物墨水。支撑浴由紧密堆积的明胶微粒等材料组成，该微粒充当宾厄姆塑料，并在打印后保持生物墨水的预期形状和结构。一旦打印的结构凝固，就可以通过将温度升高到37°C来溶解支撑浴⁷。

最近的一篇评论文章总结了使用FRESH技术在各种出版物中进行3D生物打印的材料。这些天然来源的材料范围从 I 型胶原蛋白到甲基丙烯酸化透明质酸，代表了几种不同的凝胶机制⁷。使用这种3D生物打印技术进行的大多数研究都采用静态生物材....

研究方案

1. PEGαMA的合成与表征

注：聚乙二醇-α-甲基丙烯酸酯（PEGαMA）的合成改编自Hewawasam 等人， 并在无水分条件下进行⁹。

1. 称量反应物。
   注：例如，称取 5 g 10 kg/mol 8 臂 PEG-羟基 （PEG-OH） 和 0.38 g 氢化钠 （NaH）（参见 材料表）。
2. 向 250 mL 舒伦克烧瓶中加入搅拌棒，并用氩气吹扫。
3. 将PEG-OH溶解在舒伦克烧瓶中溶解所需的最低体积的无水四氢呋喃（THF）中。
   注意：大约 80 mL 的 THF 将溶解 5 g PEG-OH。加入溶解 PEG-OH 所需的最少量 THF。
4. 向反应混合物中加入3倍摩尔过量的NaH，并在室温（RT）下搅拌30分钟。
5. 将摩尔过量的 2-（溴甲基）丙烯酸乙酯（EBrMA，见 材料表）滴加到 Schlenk 烧瓶中，并用铝箔覆盖反应容器以保护其避光。在室温下搅拌反应约48小时。
   注：对于 5 g PEG-OH 和 0.38 g NaH，使用 3.68 mL EBrMA 进行该反应。
6. 加入几滴 1 N 乙酸以淬灭反应。通过助滤剂对溶液进行真空过滤。
   注意：添加醋酸会产生气泡。当....

讨论

响应受控光照的双阶段聚合反应可以通过空间和时间控制使生物材料变硬。一些研究利用这种技术来评估各种平台中的细胞-基质相互作用 5,8,9,10,11,21,22,23。更具体地说，光掩模或双光子激发方法?.......

材料

Name	Company	Catalog Number	Comments
AccuMax Radiometer/Photometer Kit	Spectronics Corporation	XPR-3000	To measure light intensity, used for photostiffening
Acetic Acid	Fisher Scientific	BP2401-500	Used during PEGaMA synthesis
Acetone	Fisher Scientific	A184	Used with the cryosections
ActinGreen 488 ReadyProbes	Fisher Scientific	R37110	Used for staining
Aluminum Foil	Reynolds	F28028
Anhydrous Tetrahydrofuran (THF)	Sigma-Aldrich	401757-1L	Used during PEGaMA synthesis
Argon Compressed Gas	Airgas	AR R300	Used during PEGaMA synthesis
8 Arm Poly(ethylene glycol)-hydroxyl (PEG-OH)	JenKem Technology	8ARM-PEG-10K	Used during PEGaMA synthesis
365 nm Bandpass Filter	Edmund Optics	65-191	Used for photostiffening
Bovine Serum Albumin (BSA)	Fisher Scientific	BP9700-100	Used during staining process
Buchner Funnel	Quark Glass	QFN-8-14	Used during PEGaMA synthesis
Calcein AM	Invitrogen	65-0853-39	Used during staining process
Celite 545 (Filtration Aid)	EMD Millipore	CX0574-1	Used during PEGaMA synthesis
Charged Microscope Slides	Globe Scientific	1358W
Chloroform-d	Sigma-Aldrich	151823-10X0.75ML	Used to characterize PEGaMA
Click-iT Plus EdU Cell Proliferation Kit	Invitrogen	C10637	Used for staining
50 mL Conical Tubes	CELLTREAT	667050B
Cryogenic Safety Kit	Cole-Parmer	EW-25000-85
Cryostat	Leica	CM 1850-3-1
Dialysis Tubing	Repligen	132105
4’,6-Diamidino-2-Phylindole (DAPI)	Sigma-Aldrich	D9542-1MG	Used for staining
Diethyl Ether	Fisher Scientific	E1384	Used during PEGaMA synthesis
1,4-Dithiothreitol (DTT)	Sigma-Aldrich	10197777001	Bioink component
Dulbecco's Modified Eagle's Medium (DMEM)	Cytiva	SH30271.FS
Ethyl 2-(Bromomethyl)Acrylate (EBrMA)	Ambeed Inc.	A918087-25g	Used during PEGaMA synthesis
Filter Paper	Whatman	1001-090	Used during PEGaMA synthesis
Freezone 2.5L Freeze Dry System	Labconco	LA-2.5LR	Lyophilizer
Fusion 360	Autodesk	N/A	Software download
2.5 mL Gastight Syringe	Hamilton	81420	Used for bioprinting
15 Gauge 1.5" IT Series Tip	Jensen Global	JG15-1.5X	Used for bioprinting
30 Gauge 0.5" HP Series Tip	Jensen Global	JG30-0.5HPX	Used for bioprinting
Goat Anti-Mouse Alexa Fluor 555 Antibody	Fisher Scientific	A21422	Used for staining
Glycine	Fisher Scientific	C2H5NO2	Used during staining process
Hemocytometer	Fisher Scientific	1461
Hoechst	Thermo Scientific	62249	Used during staining process
Human Pulmonary Artery Adventitial Fibroblasts (HPAAFs)	AcceGen	ABC-TC3773	From a 2-year-old male patient
Hydrochloric Acid (HCl)	Fisher Scientific	A144-500	Used to pH adjust solutions
ImageJ	National Institutes of Health (NIH)	N/A	Free software download
ImmEdge® Pen	Vector Laboratories	H-4000	Used during staining process
Incubator	VWR	VWR51014991
LifeSupport Gelatin Microparticle Slurry (Gelatin Slurry)	Advanced Biomatrix	5244-10GM	Used for bioprinting
Light Microscope	Olympus	CKX53	Inverted light microscope
Lithium Phenyl-2,4,6-Trimethylbenzoylphosphinate (LAP)	Sigma-Aldrich	900889-5G	Photoinitiator used for photostiffening
Liquid Nitrogen	N/A	N/A
LulzBot Mini 2	LulzBot	N/A	Bioprinter adapted
Methacryloxyethyl Thiocarbamoyl Rhodamine B	Polysciences Inc.	669775-30-8
2-Methylbutane	Sigma-Aldrich	M32631-4L
Microman Capillary Pistons CP1000	VWR	76178-166	Positive displacement pipette tips
MMP2 Degradable Crosslinker (KCGGPQGIWGQGCK)	GL Biochem	N/A	Bioink component
Mouse Anti-Human αSMA Monoclonal Antibody	Fisher Scientific	MA5-11547	Used for staining
OmniCure Series 2000	Lumen Dynamics	S2000-XLA	UV light source used for photostiffening
Paraformaldehyde (PFA)	Electron Microscopy Sciences	15710	Used to fix samples
pH Meter	Mettler Toledo	FP20
pH Strips	Cytiva	10362010
Phosphate Buffered Saline (PBS)	Hyclone Laboratories, Inc.	Cytiva SH30256.FS
Pipette Set	Fisher Scientific	14-388-100
10 µL Pipette Tips	USA Scientific	1120-3710
20 µL Pipette Tips	USA Scientific	1183-1510
200 µL Pipette Tips	USA Scientific	1111-0700
1000 µL Pipette Tips	USA Scientific	1111-2721
Poly(Ethylene Glycol)-Alpha Methacrylate (PEGαMA)	N/A	N/A	Refer to manuscript for synthesis steps
Poly(Ethylene Oxide) (PEO)	Sigma-Aldrich	372773-250G	Bioink component
Positive Displacement Pipette	Fisher Scientific	FD10004G	100-1000 µL
Potassium Hydroxide (KOH)	Sigma-Aldrich	221473-500G	Used to pH adjust solutions
ProLong Gold Antifade Reagent	Invitrogen	P36930	Used during staining process
Pronterface	All3DP	N/A	Software download
Propidium Iodide	Sigma-Aldrich	P4864-10ML	Used for staining
RGD Peptide (CGRGDS)	GL Biochem	N/A	Bioink component
Rocker	VWR	10127-876
Rotary Evaporator	Thomas Scientific	11100V2022	Used during PEGaMA synthesis
Rubber Band	Staples	808659
Schlenk Flask	Kemtech America	F902450	Used during PEGaMA synthesis
Slic3r	Slic3r	N/A	Software download
Smooth Muscle Cell Growth Medium-2 (SmGM-2) BulletKit	Lonza	CC-3182	Kit contains CC-3181 and CC-4149 components
Sodium Hydride	Sigma-Aldrich	223441-50G	Used during PEGaMA synthesis
Sorvall ST 40R Centrifuge	Fisher Scientific	75-004-525
Stir Bar	VWR	58948-091
Syringe Filter	VWR	28145-483	Used to sterile filter solutions
T-75 Tissue-Cultured Treated Flask	VWR	82050-856	Used for cell culture work
Tissue-Tek Cyromold	Sakura	4557
Tissue-Tek O.C.T Compound (OCT)	Sakura	4583
Tris(2-Carboxyethyl) Phosphine (TCEP)	Sigma-Aldrich	C4706-2G
Triton X-100	Fisher Bioreagents	C34H622O11	Used during staining process
Trypan Blue	Sigma-Aldrich	T8154-20ML	Used for cell culture work
0.05% Trypsin-EDTA	Gibco	25-300-062	Used for cell culture work
Tween 20	Fisher Bioreagents	C58H114O26	Used during staining process
Upright Microscope	Olympus	BX63F	Fluorescent microscope capabilities
Water Bath	PolyScience	WBE20A11B
24-Well Tissue Culture Plates	Corning	3527