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What is the pH of the reconstituted HyStem components?

Gelin-S® (thiol-modified gelatin) is packaged in 5.0 mL vials containing 50mg. Vials are blanketed by nitrogen and under a slight vacuum.

We recommend using a Collagenase/Hyaluronidase mixture from Stemcell Technologies found here:

The hyaluronic acid has been thiol-modified on the carboxy groups. These active groups react with acrylate groups on PEGDA, resulting in a hydrogel.

HyStem Hydrogels are virtually transparent and should not interfere with microscopy.

Yes. Peptides that contain a cysteine residue can be used. The cysteine residue must be present for the peptide to be covalently bonded to the hydrogel substrate.

In general, the pore size for HyStem-C and HyStem-HP hydrogels is ~17 nm. In «Zarembinski, T., & Skardal, A. (2019). HyStem®: A Unique Clinical Grade Hydrogel for Present and Future Medical Applications. IntechOpen. doi: 10.5772/intechopen.81344» they use a value of

What is the shelf-life of HyStem hydrogel components?

Gelin-S ® is thiol-modified gelatin (denatured collagen) and is a component of the HyStem ® -C, and HyStem-HP hydrogel kits. Most cells do not grow well on Gelin-S–only hydrogels. Instead, Gelin-S should be used in conjunction with Glycosil ® (thiol-modified hyaluronic acid) or Heprasil ® (thiol-modified hyaluronic acid with thiol-modified heparin). Reconstituted Gelin-S remains liquid at 15 to 37°C.

Take the 10 mL bottle of Buffer A that comes with the 5 pack of glycosil 1 mL vials, and add an additional ~100-130 uL of sterile 1M NaOH to the Buffer A. Use this new Buffer A to solubilize the glycosil at 2% and you should end up with a pH of around 7.4-7.6.

The approximate molecular weight of the thiolated hyaluronic acid is 100 kDa.

One way to change the gelation time of a hydrogel is to vary the amount of crosslinker used. Gels with a lower amount of Extralink crosslinker will have a longer gelation time than those with a higher amount of crosslinker. Changing the amount of crosslinker will produce slight changes in gelation time.

Gelin-S has been thiol-modified in the same manner as the hyaluronan in Glycosil (or Heprasil), so that it covalently crosslinks with the Extralink in the HyStem hydrogels.

Trypsin, Dipase, collagenase, and hyaluronidase have been used to help detach cells from the surface or from within HyStem hydrogels.

Are any cellular attachment sites included in the HyStem hydrogel?

Please reach out through the Contact Us tab if you have further questions. We appreciate working with you.

The Buffer A is used to solubilize Glycosil, and is a buffered PBS. This will need to be adjusted, to ensure that the 2% Glycosil ends at a neutral pH.

We strongly recommend using the HyStem components the same day that they are solubilized, in order to minimize the chance of autocrosslinking. We cannot guarantee the quality of the product past the first day. If experiments need to be done across two or more days, we recommend leaving the solubilized components at room temperature overnight (capped, in the original vials). We do not recommend re-freezing the components once solubilized.

Gelation time is affected by multiple aspects of the gel’s composition.

One year from the date of receipt, if stored properly.

HyStem hydrogels may generate mild inflammation as part of the body’s natural healing process in response to injury. HyStem hydrogels do not trigger immune response when used in vivo. (These products are not for human use)

Can I concentrate Glycosil to 2% instead of just 1%?

HyStem Version 1.0 kit components were reconstituted in degassed water resulting in a pH neutral isotonic salt hydrogel. To create a more consistent and advanced product, we have made the following change:

Store Gelin-S in the original vial, unopened, at -20 °C for up to one year. Do not uncap the Gelin-S vials since they will crosslink in the presence of oxygen. Use a syringe and needle to add 1X PBS to the vial.

We routinely perform Ellmans test on our Thiolated Hyaluronic Acid. Our product falls between 0.55-0.75 umoles/mg, resulting in a degree of thiolation between 20-30%.

No. The compliance of the hydrogels is set by the amount of Extralink crosslinker added, the concentration of Glycosil (or Heprasil) and Gelin-S used, and the ratio of Glycosil (or Heprasil) to Gelin-S. Once this chemical structure of the hydrogel is fixed, it is not altered by prolonged exposure to cell culture medium.

When reconstituted using the reconstitution buffer, the pH of each HyStem component will be approximately 7.4-7.6.

Gelation time can be dramatically changed by varying the Glycosil (or Heprasil) and Gelin-S concentrations. Concentrated solutions of Glycosil (or Heprasil) and Gelin-S will create a solution with a much shorter gelation time. This can easily be done by reconstituting the components in a smaller volume of DG Water. Alternatively, diluting these components in larger volumes of DG Water will dramatically increase the total time to form the hydrogel.

How can I harvest my cells from a HyStem Hydrogel?

Possible causes are:

HyStem hydrogels and sponges differ in hydration and homogeneity. HyStem sponges are typically polymerized hydrogels that are subsequently freeze-dried. The resulting sponge is a fibrous, mesh network with pores and niches that enable cells to infiltrate and adhere. A true HyStem hydrogel is an encapsulating liquid that polymerizes around suspended cells in culture.

The gelatin used to make Gelin-S is from Type A Gelatin, Bloom 250, derived from porcine skin.

2.9. Gene expression analysis by qRT-PCR

HyStem V2.0 kit components will now be reconstituted with a buffered 1X PBS. The resulting components will also result in a pH neutral isotonic salt hydrogel. The ending pH, osmo, and rheological properties are the same from Version 1.0 to Version 2.0.

While we don’t have a specific protocol yet, we found this that might be helpful:

Can I tune the stiffness of HyStem hydrogels by altering the Extralink concentration?

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Yes. ECM proteins, such as laminin, collagen, fibronectin, or vitronectin can be non-covalently incorporated into the hydrogel prior to crosslinking.

Note: It is recommended to reconstitute each vial in its entirety.

HyStem is degraded in vivo by matrix metalloproteinases (collagenases) and hyaluronidases.

HyStem sponges can be terminally sterilized by E-beam. HyStem hydrogels have not yet been validated for use with E-beam sterilization methods. HyStem hydrogels are not terminally sterilized by gamma irradiation.

Data was collected using an Elastonsens rheometer from Rheolution: www.rheolution.com

Product Q & A

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Globular particles less than 75 kDa should be able to freely diffuse through a HyStem hydrogel.

Possible causes are:

Gelin-S provides cellular attachment sites when incorporated in the hydrogel. Gelin-S is thiol-modified, denatured collagen I, derived from either bovine or porcine sources. Gelin-S is included in all HyStem-C and HyStem-HP kits.

Yes, the Extralink concentration can be adjusted to tune the gelation speed and final gel strength of HyStem hydrogels, as shown below. The concentration represents the concentration of the extralink prior to mixing with Glycosil, not the final extralink concentration in the HyStem hydrogel. To clarify, we used a bulk bottle of Extralink https://advancedbiomatrix.com/extralink-pegda.html and solubilized that material at 0.5 — 4%. We then added that more concentrated Extralink to the other HyStem components in the same ratios as listed in the DFU.

For gene expression analysis 5 × 104 cells and 250 ng BMP-2 were encapsulated into the HyStem-HP® hydrogel and injected into the mPCL scaffold. The cells were cultured for 3 and 14 days prior to RNA isolation. Total RNA was isolated using Trizol (Life Technologies) according to the instructions provided by the supplier. RNA was quantified using a Nanodrop spectrophotometer. For cDNA synthesis, 2 μg of total RNA was used in the reverse transcription reaction. The reverse transcriptase M-MuLV (New England Biolabs) was used to synthesis cDNA according to the manufacturer’s instruction. Briefly, 1 μg of total RNA was subjected to reverse transcription using M-MuLV reverse transcriptase. The initial denaturation and priming reaction consisted of 1 μl oligo dT primer (40 μM), 1 μl random hexamer primer (40 μM), 1 μl dNTP (10 mM), 1 μg RNA, and nuclease free water to a final volume of 16 μl. After denaturation at 70 °C and priming at 4 °C, complementary DNA was synthesised by the addition of 1 μl of M-MuLV Reverse Transcriptase (200 units/μl) and 2 μl of 10× RT buffer. The reaction mixture was incubated at 42 °C for 1 h followed by enzyme deactivation at 90 °C for 10 min. The prepared cDNA was stored at −20 °C until further analysis.

What size molecules can diffuse through a HyStem hydrogel?

What is the degree of thiolation for the Glycosil?

Источники:

https://www.facebook.com/Turski.Seriali.i.Aktiori/posts/441913481271004/&rut=af65bf91f900c6ca3cf6784973b78eaa2ba11ffe1d220f5d61e3a08b148a0831
https://m.youtube.com/watch?v=mE-xZ4uQRHc&rut=8b78e2eb82e6cf722eef32ab1eb1c8376e7655f3b226b377870cae58287c04f3
https://en.wikipedia.org/wiki/Sari_Gelin&rut=1c66f50ef67fa1a670da09eb460f3e409f2405bacfa52c9c9cd411027e1f47f3
https://m.youtube.com/watch?v=T-PjFB3YiuA&rut=95ea543f2d90ab6141b66112f309887056dae136d5ce2c2f7bd9434308745cea
https://m.imdb.com/title/tt0426696/&rut=c23fb192e85dc272e0a1f819bb46a6e8403a025b1b10c5db8408d9e6b9247ef5
https://en.wikipedia.org/wiki/Fiona_G%C3%A9lin&rut=d7ca1a4a824d4762546be1d8c764a8e8da1df9a3d10764ae9621a4134e8c6be2
https://m.youtube.com/watch?v=pa322B5wqYU&rut=55286f112090c789035cc2fd9dc33c0b43b733bc72b25282cdd2cdd84fe2aa23
https://m.imdb.com/title/tt7561642/&rut=24ebe1a97991de58ffcf80b1d01ef64ee9f5c3656ea7413dd347c6d0c8e0d52f
https://advancedbiomatrix.com/gelin.html&rut=78c02f2a5c998930d43631059088c7ee91eafc6a82dde912e48c5e7e707af7af