Product Description
Advanced BioMatrix offers PhotoHA®, a methacrylated hyaluronic acid (HAMA) for photocrosslinkable hydrogels. These hydrogels provide native-like 3D HA gels with the unique attributes to be prepared at various concentrations and photocrosslinked to provide various gel stiffness. The kit comes with 100 mg of lyophilized methacrylated hyaluronic acid and ruthenium and sodium persulfate photoinitiators (400-450 nm visible light photocrosslinking).
| Item | Catalog Number | Package Size | Storage Temperature |
| Methacrylated Hyaluronic Acid | #5212 | 100 mg | -20°C |
| RutheniumPhotoinitiator | #5246 | 100 mg | Room Temperature |
| Sodium Persulfate Photoinitiator | #5247 | 500 mg | Room Temperature |
Hyaluronic acid is the most abundant glycosaminoglycan in the body being an important component of several tissues throughout the body. While it is abundant in extracellular matrices, hyaluronic acid also contributes to tissue hydrodynamics, movement and proliferation of cells, and participates in a number of cell surface receptor interactions.
For the majority of cell types, it is recommended to add additional ECM proteins to the hyaluronic acid hydrogels. The proteins provide important cell binding sites.
Storage:
The product ships on frozen gel packs. Upon receipt, store the PhotoHA®at -20°C. Store the Ruthenium and Sodium Persulfate at room temperature. The product and components are stable for a minimum of 1 year at receipt in powder form.
Once solubilized, the PhotoHA®can be stored at 2-10°C for 1 month. The photoinitiator can be stored for no more than 2 weeks once solubilized.
| Parameter, Testing, and Method | MethacrylatedHyaluronic Acid#5212 |
| Sterilization Method | Filtration |
| Sterility - USP modified | No growth |
| Form | Lyophilized Powder |
| Package Size | 100 mg |
| Storage Temperature | -20°C |
| Shelf Life | Minimum of 6 months from date of receipt |
| Shelf Life After Reconstitution | 1 Month |
| Degree of Methacrylation | 45-65% |
| Molecular Weight | 100-150 kDa |
NMR Analysis | Characteristic |
Hydrogel Young's Modulus E (Pa) | Characteristic |
Swelling Characteristics of PhotoHA®:
50 μL hydrogels fabricated in 4.7 mm diameter molds were imaged before and after incubation in phosphate buffered saline at 25°C for 24 hours. The diameter of hydrogels were quantified using ImageJ software. Statistical comparisons between groups (n=3) were performed via one-way ANOVA with post hoc testing and significance determined atp < 0.05.
Compressive Modulus of PhotoHA®:
Dynamic mechanical analysis (Q800, TA Instruments) was performed on 50 μL hydrogels fabricated in 4.7 mm diameter molds. Hydrogels were secured within a fluid cup via a 0.01 N pre-load and compressed to 30% strain at a rate of 0.5 N min-1. The Young’s modulus of each hydrogel was calculated as the slope of generated stress-strain curves between 10% and 20% strain. Statistical comparison between MeHA concentrations (n=3) was performed via Students t-test with two-tailed criteria and significance determined atp < 0.05.
Reaction Behaviorof PhotoHA®:
Rheological time sweeps (AR2000 stress controlled rheometer, TA Instruments; 0.5% strain, 1 Hz, 25°C) of MeHA crosslinking with exposure to UV light (=320-390 nm) and in the presence of 0.05 wt% Irgacure 2959 (I2959). After 1 minute, the macromer solution (i.e. MeHA and I2959) was exposed to UV light, resulting in the plateau of moduli before 5 minutes.
Directions for Use
Download the full PDF versionor continue reading below:
3D Hydrogel Preparation:
Note: Employ aseptic practices to maintain the sterility of the product throughout the preparation and handling of the collagen and other solutions.
Recommended concentrations are 5-30 mg/ml(0.5-3.0%).
Note: The following recommended instructions are for a 1% hyaluronic acid (HA) methacrylate solution. Adjustments to this protocol may be required for various concentrations.
- Add 10 ml of 1X phosphate buffer saline (PBS), water or cell culture media to the 100 mg of lyophilized methacrylated HA powder.
- Mix on a shaker table or rotator plate until fully solubilized (~30 to 60 minutes) at 2-10°C.Note: Solubilization times may vary depending on the desired concentration and volume of PBS, water or medium added.
- Calculate the volume of photoinitiator to add by multiplying the volume of solubilized hyaluronic acid by 0.02. If the resulting number is 200 ul, for example, you will add 200 ul of ruthenium and 200 ul of sodium persulfate.
- Solubilize the required amount of ruthenium (per step 3) at a concentration of 37.4 mg/ml in 1X PBS or cell culture media.
- Solubilize the required amount of sodium persulfate (per step 3) at a concentration of 119 mg/ml in 1X PBS or cell culture media.
- Add the ruthenium to the hyaluronic acid solution and fully mix until solution is homogeneous.
- Add the sodium persulfate to the hyaluronic acid/ruthenium solution and mix until solution is homogeneous.
- Add your cells to the hyaluronic acid/photoinitiator solution.
- Dispense your hyaluronic acid/photoinitiator/cell solution into the desired dish (ie. 6-well plate, 48-well plate).
- For photocrosslinking, place solution directly under a 400-450 nm visible light crosslinking source.
Product Q & A
Yes. There are quite a few publications citing various hyaluronidase protocols for digesting PhotoHA hydrogels. Here are a few:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460858/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4840832/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986518/
The HA we usecomes frombiotechnological production, where the hyaluronic acid is extracted from the cell walls of the bacteria Streptococcus zooepidemicus.
Product Cell Assay
Human mesenchymal stem cells (20 x 106 / mL) were encapsulated in 50 μL hydrogels (~ 4.7 mm x 2 mm). Hydrogels (1 wt% PhotoHA®) were fabricated with 0.05 wt% Irgacure 2959 and exposure to 2 mW/cm2 light (320-390 nm) for 10 minutes. After 24 hours, encapsulated cells were stained with calcein AM and ethidium homodimer and subsequently imaged on a Leica SP5 confocal microscope (using FITC/TRITC sequential scans).
Product Applications
PhotoHA® Methacrylated Hyaluronic acid can be used to form hydrogels for ex-vivo engineering of autologous cartilage tissue[1] or as a mesenchymal stem cell carrier in cartilage repair[2].
Because the stiffness can be widely adjusted by altering concentration or UV-light exposure, methacrylated HA has been used to measure the effects of matrix stiffness on cell phenotype and function[3][4].
Methacrylated HA can be used for 3D bioprinting (extrusion[5], inkjet[5] and photolithographic[6]) to create structures that promote osteogenic differentiation of MSC’s[7].
The high tunability of hyaluronic acid methacrylate allows it to be mixed with, and reinforce other types of hydrogels (such as collagen, or gelatin methacrylate)[8].
References:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717235/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627486/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541838/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447944/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615317/
https://www.ncbi.nlm.nih.gov/pubmed/21773726/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460858/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748291/
Product References
References for PhotoHA®:
Khetan, Sudhir, and Owen Corey. "Maintenance of stem cell viability and differentiation potential following cryopreservation within 3-dimensional hyaluronic acid hydrogels."Cryobiology(2019).
Poldervaart, M. T.et al.3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity.Plos One12,(2017).
Product Certificate of Analysis
Safety and Documentation
Safety Data Sheet
Certificate of Origin
Product Disclaimer
This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
美国AdvancedBioMatrix（简称ABM） www.advancedbiomatrix.comAdvancedBioMatrix（简称ABM）是美国一家著名的生物公司，获得了AllerganInc的授权(Allergan用25年时间不断完善胶原蛋白相关的产品的生产工艺)，将Allergan的专业和技术用于蛋白生产与检测，致力于为组织工程、细胞分析及细胞增殖等研究领域提供优质稳定的产品。AdvancedBioMatrix不断丰富已有产品线，目前可为三维细胞培养提供各种胶原蛋白、纤连蛋白、玻连蛋白、水性凝胶、不同粘度与分子量的透明质酸以及低代成纤维细胞等。在美国全部产品授权Sigma销售。AdvancedBioMatrix是组织培养，细胞分析和细胞增殖三维（3D）应用的生命科学领域的领导者。我们的产品被公认为纯度，功能性和一致性的标准。我们在生产，分离，纯化，冷冻干燥，细胞培养和蛋白质测试，粘附肽，附着因子，底物刚性和其他3D矩阵产品方面拥有丰富的专业知识。我们的专业技术和知识正在被用来确保我们的产品质量最高，批次之间一致且易于为我们的研究客户使用。
美国AdvancedBioMatrix是3D组织培养、细胞检测和细胞增殖等领域实验解决方案的佼佼者。AdvancedBioMatrix在分离、纯化、冻干、细胞培养和蛋白检测、多肽粘附、附着因子、基质硬度和其他3Dmatrix 产品开发方面有着丰富的经验。AdvancedBioMatrix的研发经验和专业知识确保其产品可达到最佳质量，并保证产品之间一致性，方便研究客户使用。以下为AdvancedBioMatrix3DMatrices 产品竞争优势：1. 提供高纯度和成分确定的胞外基质；2. 超过1000余篇文献引用PureCol产品，品质非常均一；3. 在3D培养基领域可提供最全面的产品线；4. 唯一可提供特异性刚性有机硅基板的公司（CytoSoft）;5. 唯一可提供可溶性丝纤蛋白的供应商（可运用于多种3D培养）；6. 如果客户首次接触3D胶原凝胶，AdvancedBioMatrix还是唯一的预制胶原蛋白(PureColEZGel)供应商；
以下产品为AdvancedBioMatrix全球畅销品：1.PureCol 牛源I型胶原蛋白 3mg/ml#5005-100ML2.Nutragen牛源I型胶原蛋白 6mg/ml#5010-50ML3.FibriCol 牛源I型胶原蛋白 10mg/ml#5133-20ML4.VitroCol 人源I型胶原蛋白 #5007-20ML5. 弹性蛋白原 #5052-1MG6.ECMSelectArraykitUltra-36#5170-1EA7.CytoSoft（刚性可变的基底，AdvancedBioMatrix最新添加产品5190-7EA）8. 人III型胶原蛋白 #5021-10MG9. 人IV型胶原蛋白 #5022-5MG10.SilkFibroin溶液 #5154-20ML11.Fibronectin#5080-5MG12.Vitronectin#5051-0.1MG
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最近开始做TUNEL染色，第一次按罗氏protocol做的，阴性对照是没有显色的。没做阳性对照。第二次、第三次做出来背景都很深，肾小管每个细胞核上都染上了棕黄色，DAB染色很快，几秒就变一片黄。具体步骤是：1烤片脱蜡至水2.蛋白酶K20ug/ML37℃10分钟3.tunel液冰上配置后加组织上，37度1h。4.pod液37℃30min5.DAB显色。在蚂蚁淘中检索后，优化了实验条件，在蛋白酶K前加了3%H2O2封闭10min，缩短了tunel液至40min。这是第三次的结果大家帮我看看
这是对照组
这是模型组
Bone Mesenchymal Stem Cells 作为一个细胞群体，还没有发现有特定细胞表面marker. 对于那些可以代表自我更新和分化的marker, 也不清楚到底要发现哪一个的表达才能确定该细胞就是BMSC。
目前常用的方法，就是采用培养，colony-forming unit-fibroblasts (CFU-F)这个方法。一般BMSC可以24-48小时贴壁。
流式细胞计数，比如STRO-1，但是一般认为STRO-1阳性的细胞更趋向于造血干细胞，和BMSC简单区别还不是很清楚。
这里有个培养分化的产品
http://www.rndsystems.com/pdf/SC020.pdf
