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|
| Q. |
What cell types/applications are suitable for this material? |
| A. |
PuraMatrix™ Peptide Hydrogels, including the related
family of amphiphilic peptide gels, have been shown to promote the differentiation
of primary rat hepatocytes and hepatocyte progenitor cells, 1 murine endothelial and cardiomyocyte cells, 2&3 rat pheochromocytoma cells (PC12), 4 and hippocampal neurons. 5 Studies
have also demonstrated that PuraMatrix™ Peptide Hydrogel
supports the attachment of a variety of primary (e.g., neuronal, fibroblast,
keratinocyte) and transformed (e.g., MG-63, SH-SY5Y, HEK293, NIH3T3) cell
types. 6 A partial list of other
applications in which PuraMatrix has been used include:
* Hepatocyte toxicity, induction and metabolism assays
* Stem cell proliferation and differentiation assays
* Tumor cell migration and invasion
* Angiogenesis assays
* Cardiomyocyte cell culture and in vivo delivery
* Neurite outgrowth assays
* Co-culture of various cell types* Osteoblast bone growth
* Chondrocyte & cartilage culture
* Endothelial, neural, osteoblast 3-D cell structure formation
* Tissue models for in vitro drug screening
* Tissue engineering research
* Bioproduction using PuraMatrix microcarriers in suspension culture
* In vivo analyses of tissue regeneration |
|
| Q. |
Does PuraMatrix™ Peptide Hydrogel promote cell growth and differentiation in the absence of protein/growth factor supplementation? |
| A. |
Often, PuraMatrix™ can complement the typical culture
media additives and substitute directly for other matrices, substrates
and scaffolds. To achieve optimal cell growth and differentiation, it
is necessary to determine the appropriate mixture of PuraMatrix™
Peptide Hydrogel and bioactive molecules (e.g., growth factors, ECM proteins,
and/or other molecules). |
|
| Q. |
Should cells be plated on top of the hydrogel (2D surface plating) or encapsulated within the material (3D encapsulation protocol)? |
| A. |
Either option is possible. The optimal plating configuration will
depend on the cell type and the experimental objectives. Please refer
to the Guidelines for Use for specific examples and recommendations. |
|
| Q. |
If cells are seeding onto the material using the surface plating method, will the cells migrate into the hydrogel? |
| A. |
Since the hydrogel 'fibers' are flexible (not covalently linked),
certain cells will migrate into the hydrogel. 3-D cell structure formation
(spheroids, endothelial tubes, neuronal networks, stromal co-cultures,
etc.) will depend on the cell types and culture media composition. The
capacity for migratory behavior will be dependent on the cell type used. |
|
| Q. |
What seeding density should be used? |
| A. |
We suggest a seeding density comparable to that used for surface
plating on standard tissue culture-treated or ECM-coated substrates. A
seeding density of 0.5-1.0 x 10 6 cells/ml is recommended for 3D encapsulation
cultures. |
|
| Q. |
PuraMatrix™ gels seem too thick? How can I best seed cells, proteins and other additives into the gels with an even distribution? |
| A. |
PuraMatrix™ thickens after sitting in the vial
over a period of a few hours, but this gel can be liquified (prior to
the addition of salt or media) by sonication, vortexing, or even pipetting
up and down. |
|
| Q. |
How long can cells survive on PuraMatrix™ Peptide Hydrogel? What about scaffold degradation? |
| A. |
The actual duration of the culture will depend on the cell type
and culture conditions, but cultures in PuraMatrix has been taken out
for weeks and months. PuraMatrix scaffolds can last for many weeks in
culture and dependes on the concentration used as well as the cell type. |
|
| Q. |
Can I sub-culture cells that have been maintained on PuraMatrix™ Peptide Hydrogel? |
| A. |
Yes. Cells can be recovered from the hydrogel and then sub-cultured for an additional period of growth/differentiation using a fresh layer
of PuraMatrix™ Peptide Hydrogel or an alternative growth
substrate (e.g., TC-treated, ECM-coated). Please refer to the Guidelines
for Use for the cell recovery protocol. |
|
| Q. |
Can I co-culture cells using PuraMatrix™ Peptide Hydrogel? |
| A. |
Co-culturing of two different cell types can be achieved by first encapsulating the first cell type in PuraMatrix™ and then overlaying
the second cell type on the layer containing the first cell type. PuraMatrix
matrices can be used in a similar manner to collagen or Matrigel ECM gels
in sandwich cultures. |
|
| Q. |
How is PuraMatrix™ similar and/or different from agarose, collagen, hyaluronic acid,poly(ethylene glycol), and methycellulose hydrogels
for long-term cultures? |
| A. |
PuraMatrix™ is similar to agarose, collagen and methylcellulose
in that it will typically support the long-term growth of many different
cell types, either for differentiation or tumorigenicity studies. Unlike
collagen substrates and scaffolds, PuraMatrix will not shrink due to contraction
by fibroblasts and other cell types.
PuraMatrix™ is different from the three listed matrices
in that it is transparent, synthetic and supports anchorage dependent
cell types. PuraMatrix is often used at much lower concentrations (i.e.
1.0%, 0.5% and 0.25%) than other hydrogels, improving nutrient diffusion,
cell morphology and cell viability. PuraMatrix forms a loose gel, allowing
improved culture, complex cell structure formation, analysis and recovery
of cells. In many cases, differential cell growth will be evident and
it is much easier to isolate cells, DNA and proteins from PuraMatrix™
than other synthetic and animal-derived matrices. |
|
| Q. |
How do I add extracellular matrix proteins (Laminin, Collagen I, Collagen IV, Fibronectin, Integrin sequences, etc.) into PuraMatrix™
to create defined ECMs? |
| A. |
PuraMatrix™ can be mixed with ECM proteins, growth factors,
and other additives. Often, these additives are packaged in solutions
containing salt, which will cause the formation of the PuraMatrix nanofibers
and create a more lumpy gel. We suggest washing the additives with a sucrose
solution to remove the salt, then mixing the additive/s with PuraMatrix
before coating plates or inserts with the mixture. |
|
|
| Q. |
Is PuraMatrix™ Peptide Hydrogel compatible with confocal microscopy and other imaging technologies? |
| A. |
Yes. This material forms a clear gel that allows good resolution
for microscopy and radiology applications. |
|
| Q. |
I am having a hard time visualizing PuraMatrix™ because it is so transparent? How can I stain PuraMatrix™? |
| A. |
PuraMatrix is quite hard to see, lending many benefits to visualizing the cells and structures growing within the scaffolds. Trypan blue can
be used to visualize the scaffolds along with dead cells, which take up
the blue stain. |
|
| Q. |
Can I perform molecular analyses of proteins and nucleic acids using cells cultured in PuraMatrix™ Peptide Hydrogel? |
| A. |
Yes. Cells cultured in the hydrogel can be prepared for most molecular techniques. Following mechanical disruption of the gel, the cells can be isolated using
centrifugation and then processed according to standard procedures. |
|
| Q. |
Is PuraMatrix™ Peptide Hydrogel compatible with staining and immunodetection protocols? |
| A. |
Yes. Cells can be stained with fluorescent dyes and most immunological reagents using standard methods. |
|
|
|
| Q. |
Why is it necessary to work quickly when mixing cells with this material prior to gelation? |
| A. |
The stock solution of PuraMatrix ™ Peptide Hydrogel (1% w/v) exhibits
a pH at 3.0, which can adversely affect cell viability. Therefore, it is
important to work quickly to minimize the amount of time that cells are in
contact with this material prior to the addition of culture medium. The culture
medium is changed three times within the first 30 minutes to equilibrate the
sample to physiological pH. |
|
| Q. |
What is the mechanical strength of PuraMatrix™ Peptide Hydrogel? |
| A. |
At the typical working concentration (0.5% w/v), the hydrogel forms a soft fibrous network that exhibits a relatively weak mechanical strength
with important benefits for cell migration, spheroid formation and angiogenesis.
Therefore, it is necessary to handle the material very carefully when
performing medium changes (i.e., avoid direct contact with the hydrogel
when using pipet or aspiration tips). Importantly, the 0.5% hydrogel has
been found to promote the attachment and growth of many cell types. To
prepare a hydrogel with greater mechanical strength, the material can
be used in the undiluted form (1% w/v). |
|
| Q. |
What is the best way to remove air bubbles that result from mixing the material with cells, bioactive molecules, and/or medium? |
| A. |
Air bubbles can be removed by sonication in a bath sonicator for 30 minutes or by centrifugation (e.g., spin two to five minutes at 5K in a tabletop centrifuge).
If small volumes are used in a 1.5 ml eppendorf tube, centrifugation can be
performed using an eppendorf microfuge for 10-15 seconds at full speed. |
|
| Q. |
Since the peptide sequence of PuraMatrix™ Peptide Hydrogel is similar to RGD, do cells bind to the material via RGD-dependent integrin
receptors? |
| A. |
No. The peptide sequence promotes cell attachment, but does not mediate RGD-dependent integrin signaling. Studies have shown that cell attachment
is not inhibited by RGD-containing peptides. |
|
|
|
| Q. |
Can PuraMatrix™ Peptide Hydrogel be used for in vivo studies in animals? |
| A. |
Yes. The soluble material can be injected and will subsequently form
a 3D hydrogel upon contact with the physiological environment. PuraMatrix™
Peptide Hydrogel can be readily handled using small/large bore needles
and catheters.
PuraMatrix can form a synthetic temporary scaffold to localize injected
cells for tumor and cancer studies. Similar to Matrigel plug assays, cells
can be seeded into PuraMatrix prior to injection -- be sure to minimize
the time cells spend in low pH PuraMatrix prior to injection -- then injected
into a tissue compartment to form a bolus of cells. |
|
| Q. |
Can PuraMatrix™ be used with fine gauge needles and catheters? |
| A. |
Yes, PuraMatrix gels remain injectable both before and after assembly of the nanofibers and are compatible with surgical methods of injection
across applications. To avoid the introduction of bubbles in vivo,
use extreme care when filling and injecting samples with needles that
are smaller than 20G. |
|
| Q. |
How can PuraMatrix™ be used in vivo? |
| A. |
PuraMatrix™ Peptide Hydrogel has been used for animal studies to complement and improve medical device implant / tissue interface
healing, wound healing, cancer/tumor implants, drug delivery and therapeutic
stem cell delivery for regenerative medicine. PuraMatrix solution will
gel to form a nanofibrous matrix upon injection in vivo, through beta-sheet
self-assembly. This resulting gel serves as a synthetic, transparent and
permissive scaffold for wound healing, tissue regeneration, drug delivery
and therapeutic cell delivery. The materials can be buffered prior to
in vivo administration to counter its low pH. |
|
| Q. |
How can PuraMatrix™ be buffered to mitigate its low pH? |
| A. |
PuraMatrix™ Peptide Hydrogel can be buffered and/or assembled into it's nanofibrous gel prior to injection to bring the pH into the
physiological range. Phosphate Buffered Saline is the best solution to
add. Note that this will cause the nanofibers to assemble prior to injection,
but the gels will still be injectable through fine gauge needles. |
|
|
|
1 Semino, C.E., et al., Functional differentiation of hepatocyte-like
spheroid structures from putative liver progenitor cells in threedimensional
peptide scaffolds. Differentiation 71:262 (2003).
2 Davis, M.E., et al., Injectable Self-Assembling Peptide
Nanofibers Create Intramyocardial Microenvironments for Endothelial Cells. Circulation 111:442-450 (2005).
3 Narmoneva, D.A., et al., Endothelial Cells Promote Cardiac
Myocyte Survival and Spatial Reorganization: Implications for Cardiac
Regeneration. Circulation 110: 962-968 (2004).
4 Holmes, T.C., et al., Extensive neurite outgrowth and
active synapse formation on self-assembling peptide scaffolds. PNAS
USA 97 :6728 (2000).
5 Semino, C.E., et al., Entrapment of migrating hippocampal
neural cells in 3D peptide nanofiber scaffold. Tissue Engineering
10:643 (2004).
6 Zhang, S., et al., Self-complementary oligopeptide matrices
support mammalian cell attachment. Biomaterials 16:1385 (1995).
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Not for resale. |
