Culturing Cells in Defined 3-D Structures

Media Supplements Matter

There have been several publications referencing use of our growth factors in 3-D Cultures. It is important that potent growth factors are used to ensure proper cell growth and differentiation.

Here's a new publication referencing use of our ISOKine^TM FGF. Our ISOKine growth factors are produced in the endosperm tissue of barley grain (Hordeum vulgare), that exhibits up to 50 times less protease activity than E.coli or mammalian cells. Barley seed is void of any human or animal viral contaminants that could jeopardize your cell culture.

Claas Willem Visser, Tom Kamperman, Lisanne P. Karbaat, Detlef Lohse and Marcel Karperien. In-air microfluidics enables rapid fabrication of emulsions, suspensions, and 3D modular (bio)materials. Science Advances 31 Jan 2018: Vol. 4, no. 1, eaao1175 DOI: 10.1126/sciadv.aao1175. In this study, the authors present in-air microfluidics (IAMF), a new chip-free platform technology that enables in-flight (that is, on-the-fly) formation of droplets, fibers, and particles and their one-step deposition into 3D constructs with a modular internal architecture.

Figure: Concept of IAMF and guide to the article. (A) Chip-based microfluidics enables in-line control over droplets and particles, making it a versatile platform technology. A chip design where droplets (blue) are transported by a coflow (pink) is shown. (B) IAMF maintains the in-line control of chip-based microfluidics but relies on jet ejection and coalescence into air. Therefore, a wide range of droplets and particles can be produced at flow rates typically two orders of magnitude higher than with chip-based microfluidics. When combining reactive, solidifying microjets, IAMF also enables on-the-fly production and direct deposition of microparticles into 3D multiscale modular (bio)materials.

Figure: One-step additive manufacturing and injection molding of 3D multiscale modular (bio)materials. (A) Modular free forms with a controlled microarchitecture were manufactured by stacking of shape-stable core-shell particles. (B to D) A hollow cylinder was formed by deposition of the composite jet onto a rotating substrate. By altering the building blocks’ composition, the resulting microarchitecture consisted of (C) a liquid-filled foam or (D) a multimaterial modular solid, where the cross-linker for the core was added to the shell and vice versa. (E) To eject a modular filler, only the droplets’ cores are solidified in the air, whereas the slower solidifying shells enable seamless filling of the mold. (F to H) A modular construct was produced by filling a bone-shaped mold. Inset: Hydrogel construct while still in the mold. The 3D multiscale modular material consisted of MSCs (pink), encapsulated in alginate microspheres (green) that are embedded in dextran-tyramine hydrogel (red). (I) Injection-molded multiscale modular tissue construct with optimized cellular micro- and macroenvironments. The construct consisted of insulin-producing pancreatic β cells (MIN6; beige with blue nuclei) that were encapsulated in alginate microparticles (green). The cell-laden microparticles were encapsulated within a proangiogenic fibrin network that contained human endothelial and stem cells (pink with blue nuclei). The microenvironments supported MIN6 cell proliferation, whereas the macroenvironment supported the formation of an endothelial cellular network within 7 days of in vitro culture. HUVEC, human umbilical cord endothelial cell. Scale bars, 1 cm (B and F), 5 mm (G), and 100 μm (C, D, H, and I).

We live in a 3-D world and 3-D Cell and Tissue Based Assays are a major focus for us. This includes bioinks for 3-D printing.

Neuromics' FGF-2 for Expanding Human Cells

FGF-2 and Fibrochondrocytes

FGF-2 of FGF-basic is an important a growth factor for many cell-based assays. Neuromics' has rock solid FGFs for supplementing media used to grow cells. Check out publications referencing use of FGF-2.

Here's the latest publication: Yan Liang, Enaam Idrees, Stephen H. J. Andrews, Kirollos Labib, Alexander Szojka, Melanie Kunze, Andrea D. Burbank, Aillette Mulet-Sierra, Nadr M. Jomha & Adetola B. Adesida. Plasticity of Human Meniscus Fibrochondrocytes: A Study on Effects of Mitotic Divisions and Oxygen Tension. Scientific Reports 7, Article number: 12148 (2017) doi:10.1038/s41598-017-12096-x. ...Thereafter the number of viable MFCs were counted using a haemacytometer after trypan blue staining. MFCs were plated at 104 cells/cm2 and cultured in the standard medium described above supplemented with FGF-2 (5 ng/mL; Neuromics, MN, USA, Catalog#: PR80001) and TGFβ1 (1 ng/mL; ProSpec, NJ, USA, Catalog#: cyt-716) under normal oxygen tension (21% O2) at 37 °C in a humidified incubator...

Images: Immunofluorescence analysis of collagen I and collagen II in pellets derived from T1F2-expanded MFCs of four passages after 21 days chondrogenic stimulation under NRX or HYP from one representative donor (male, 20 years old). Blue (DAPI): cells, Red (Texas Red): collagen I, Green (FITC): collagen II. (A) Pellets cultured under NRX, (B) Pellets cultured under HYP from four passages. Scale bar: 100 µm

Our ISO-Kine FGF-2 is especially potent as it virtually endotoxin free.

Neuromics' ISOKine bFGF in 3-D Cultures

Works Well in Perfusion Models

Our ISOKine^TM FGF is produced in the endosperm tissue of barley grain (Hordeum vulgare), that exhibits up to 50 times less protease activity than E.coli or mammalian cells. Barley seed is void of any human or animal viral contaminants that could jeopardize your cell culture.

It is a proven solution for all cell cultures and starts at the low price of  65 USD/10 ug.

Here's a reference of its use in 3-D Cultures: Tom Kamperman, Sieger Henke, Claas Willem Visser, Marcel Karperien, Jeroen Leijten. Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape. DOI: 10.1002/smll.201603711.

Figure: Delayed on-chip crosslinking enables centering of single cells in microgels. a) Fluorescence confocal imaging confirmed that delayed enzymatic crosslinking enabled centering of single MSCs in Dex-TA microgels. b) On average, cell-laden microgels were only 9 µm larger than the encapsulated MSCs, effectively resulting in 3D hydrogel coatings of less than 5 µm. c) A standard microfluidic droplet generator was connected to the H2O2 diffusion-based crosslinking chip. The position of cells (white arrows) in non-crosslinking microgel precursor droplets was analyzed d) immediately after droplet generation (t1), at the start of the crosslinking chip (t2), and e) at the end of the crosslinking chip (t3). f) Cell positions within microgels produced using conventional microfluidic encapsulation systems (i.e., with coupled emulsification and gelation) are indicated with gray (i.e., references) and red (i.e., this work) data points. Cell positions within gel precursor droplets along the modular microfluidic setup are indicated with blue data points. Cell positions within delayed enzymatically crosslinked microgels are indicated with green data points. g) Cell position analyses of various combinations of distinct hydrogel materials (i.e., Dex-TA, Dex-HA-TA, PEGDA), cell types (i.e., MIN6, MSC), and crosslinking methods (i.e., enzyme-based and photo-crosslinking), revealed that delayed crosslinking consistently resulted in significantly increased cell-centering as compared to the conventional encapsulation approach where emulsification and gelation are coupled.

If you are looking for competitively priced, animal free and potent growth factors. check out our ISOkines.

Culturing Stem Cells in 3-D

Requires Potent Media + Supplements

Neuromics is responding to the many challenges our clients face in building 3-D, in-vivo like, cell- based assays. We do this by offering the most potent Media plus Supplements like FGFS.

Here's a protocol for single cell 3-D assays using hMSCs and Hydrogels. It features use of our ISOKine^TM FGF

Images: Cell-centering in cytocompatible microgels enables long-term single-cell 3D culture by preventing cell escape. a) Qualification of Dex-TA microgel crosslinking as a function of the microemulsion flow rate (Qemulsion) and concentration of the H2O2 feed ([H2O2]feed). Blue, green, and red indicate incomplete crosslinking, complete crosslinking, and H2O2 excess, respectively. b,c) Amplex Red assay to quantify the concentration of residual H2O2 ([H2O2]emulsion) in Dex-TA microgel precursor droplets and crosslinked microgels after their retrieval from the diffusion-based crosslinking platform. d) The microencapsulation procedure had no detrimental effect on short-term cell survival. e) Delayed crosslinking resulted in 4 ± 1% cell escape after 7 d of in vitro culture, as compared to 27 ± 5% cell escape when using coupled emulsification and gelation. f) The number of encapsulated cells per microgel tightly followed the Poisson distribution and remained similar throughout long-term (28 d) of in vitro culture, which confirmed that cell centering prevents cell escape. g–i) MSCs encapsulated in delayed enzymatically crosslinked microgels remained viable and metabolically active throughout 28 d of in vitro culture. j) Positive Oil Red O and k) Alizarin Red staining confirmed that l) more than 60% of the microencapsulated MSCs could differentiate into the adipogenic and osteogenic lineage, respectively. Black scale bars: 50 µm, white scale bars: 5 µm. DOI: 10.1002/smll.20160371.

Protocol for Cell Isolation and Expansion: Human MSCs were isolated from fresh bone marrow samples and cultured as previously described. The use of patient material was approved by the local ethical committee of the Medisch Spectrum Twente and informed written consent was obtained for all samples. In short, nucleated cells in the bone marrow aspirates were counted, seeded in tissue culture flasks at a density of 500 000 cells cm−2, and cultured in MSC proliferation medium, consisting of 10% FBS, 100 U mL−1 penicillin, 100 mg mL−1 streptomycin, 1% GlutaMAX, 0.2 × 10−3 m ascorbic acid, and 1 ng mL−1 bFGF (added fresh) in αMEM. Mouse insulinoma MIN6-B1 cells (provided by Dr. P. Halban, University Medical Center, Geneva, Switzerland) were cultured in MIN6 proliferation medium, consisting of 10% (v/v) FBS, 100 U mL−1 penicillin, and 100 mg mL−1 streptomycin, and 71 × 10−6 m 2-mercaptoethanol (added fresh) in DMEM. When cells reached near confluence, the cells were detached using 0.25% Trypsin-EDTA at 37 °C and subsequently subcultured or used for experimentation.

I am at your beck and call to answer questions on our Cell Based Assay Solution. Pete Shuster-CEO and Owner, direct phone: (612) 801-1007 or pshuster@neuromics.com.

Growth Factors for Excellent Cultures

Quality Counts
Cells are the foundation of Cell Based Assays. We provide High Quality Growth Factors to insure your success.  Here's a sampling of the categories available:

Angiogenic Growth Factors
Bone Morphogenetic Proteins (BMPs)
Brain Derived Neurotrophic Factors (BDNFs)
Ephs/Ephrins
Epidermal Growth Factors (EGFs)
Fibroblast Growth Factors (FGFs) 
Hematopoietic Cell Growth Factors 
Hepatocyte Growth Factors 
Insulin-Like Growth Factors (IGFs)
Neuron-Glial Expressed
Platelet Derived Growth Factors (PDGFs)
Transforming Growth Factors (TGFs)
Vascular Endothelial Growth Factors (VEGFs)

Images: GFP+ mMN Mouse Motor Neurons. Protocol includes addition of  Neuromics' Bioactive GDNF Recombinant Protein to media for expansion/differentiation.
We will continue to add new pubs and data on these as they become available.

Bioactive FGF Basic Recombinant Protein in Action

Maintaining Sheep Mesenchymal Stem Cell Multipotency

We have been very selective in the Bioactive FGF2 or FGF-basic Recombinant Proteins we make available to Stem Cell Researchers. They are many options available so we are always encouraged when we see ours referenced in publications.

Here researchers used our FGF2 to expand Sheep Bone Marrow Stem Cells (BMSCs) in culture: Troy D Bornes, Nadr M Jomha, Aillette Mulet-Sierra and Adetola B Adesida. Hypoxic culture of bone marrow-derived mesenchymal stromal stem cells differentially enhances in vitro chondrogenesis within cell-seeded collagen and hyaluronic acid porous scaffolds.Stem Cell Research & Therapy 2015, 6:84 doi:10.1186/s13287-015-0075-4.

These cells were used for chondrogenesis studies.

Expansion of BMSCs: Bone marrow aspirate collections containing 8 x 10⁷ MNCs were seeded within each 150-cm2 tissue culture flask. Culture medium composed of alpha-minimal essential medium (α-MEM) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS), penicillinstreptomycin-glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and sodium pyruvate (all from Life Technologies, Burlington, Canada) was pipetted into each flask. Fibroblast growth factor-2 (FGF-2; Neuromics Inc., Edina, USA) was added at a concentration of 5 ng/ml in order to maintain cell multipotency. Nucleated cells were allowed to adhere and grow for seven days before the first media change under normoxia (ambient 21% O2) or hypoxia (low 3% O2) at 37°C in a humidified incubator containing 5% CO2. Flasks from the hypoxic incubator experienced short periods (less than 5 minutes) of normoxic exposure during media changes. Thereafter, the media were changed twice per week until 80% cell confluence was obtained. Adherent BMSCs were detached using 0.05% w/v trypsin-ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich Corp., Oakville, Canada) and expanded under the same oxygen tension (normoxia or hypoxia) as during isolation until P2 prior to scaffold seeding. Hereafter for brevity, BMSCs described by expansion oxygen tension alone (normoxia-expanded and hypoxia-expanded BMSCs) will refer to BMSCs that were isolated and expanded under normoxia and hypoxia, respectively. The time taken from plating of nucleated cells (P0) to reach approximately 80% confluence at P2, before experimental use, varied from three to four weeks.

I anticipate more publications on these important bioactive reports as demand for them has been growing.

The Power of ISOkine Stem Cell Growth Factors

What Endotoxin, Animal and Serum Free Means to You

ISOkine™ growth factors are produced in barley, bypassing the use of bacterial or animal cell systems.

Why Does this Matter? Proteins produced in bacterial systems contain trace Endotoxins (toxins produced by the bacterial hosts). Studies show that these Endotoxins can compromise health of your stem cell based assays. Animal and human cell expression systems pose risk to your assays, because they may harbor pathogens.

They are Low Cost and Work in the Hands of our Customers: "The ISOKine mouse LIF is an excellent product! I enjoyed dealing with Neuromics. My interactions with Brett were very professional and he assured me the product was guaranteed to work; which it did." Andy Babwah, Children’s Health Research Institute

Product Options:

Name	Catalog #	Type	Species	Size	Price
ISOKine-EGF	PR80002-100	Protein	H	100 ug	$99
ISOKine-Flt3-Ligand, human-NEW	PR80004-10	Protein	H	10 ug 100 1 mg	$129 $529 $2,649
ISOKine-bFGF	PR80001-10	Protein	H; M	10 ug 50 ug 100 ug	$65 $169 $225
ISOKine-Leukemia Inhibitory Factor (LIF), human-NEW	PR80003-10	Protein	H	10 ug 100 ug 1 mg	$90 $395 $2,475
ISOKine-Leukemia Inhibitory Factor (LIF), mouse-NEW	PR80000-10	Protein	M	10 ug 50 ug 100 ug 1 mg	$90 $250 $395 $2,475
ISOKine-SCF, human-NEW	PR80005-10	Protein	H	10 ug 100 ug 1 mg	$90 $395 $2,475

Should have questions do not hesitate to call me directly 612-801-1007 or pshuster@neuromics.com. Pete Shuster, CEO and Owner.

100% Animal Free FGF Basic

New ISO-kine^TM FGF basic-only 39 USD-10 ug.

Fibroblast Growth Factor (FGF) Recombinant Proteins are widely used as tools in Stem Cell Research. They have the capabilities to catalyze growth and differentation as well as maintain cell stasis. We continue to offer new FGFs to make sure we meet the unique demands of our cutsomers and collaborators  .

Our E. Coli derived Human FGF (146 aa) recombinant protein is our #1 seller. As Stem Cell research moves from the bench top to the bedside, there will be a growing need for 100% animal free proteins. We are please to announce we have added a 100% animal free FGF (146 aa) basic that has bio-activity comparable to our top selling options

Image: ISOKine^TM bFGF vs our potent and proven e-coli derived bFGF.

I anticipate offering more ISOKine^TM Bio-risk free Stem Cell Research growth factors to meet growing demand. This growth will be driven by their unique advantages. These include low protease activity and secondary metabolite content and simple protein content all of which aid in downstream processing. These products also have the G.R.A.S. (Generally Recognised As Safe) status from FDA.

FGF Basic and Cell Proliferation in Endothelial Cells

We are pleased with the growth of our Fibroblast Growth Factor Proteins.

Here's a recent publications referencing use of our FGF basic in Endothelial Cell Cultures.

Harun Elmasri, Cagatay Karaaslan, Yaroslav Teper, Elisa Ghelfi, MeiQian Weng, Tan A. Ince, Harry Kozakewich, Joyce Bischoff, and Sule Cataltepe. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells. FASEB J, Jul 2009; doi:10.1096/fj.09-134882

...VEGF-A 165 (VEGF) and human basic FGF (bFGF) were purchased from R&D Systems (Minneapolis, MN, USA) and Neuromics (Edina, MN, USA), respectively...

Featured Reagent
FGF basic (146 aa)

Related Reagents:
Proteins
FGF basic (146 aa), carrier-free
FGF basic (157 aa)
FGF basic (157 aa), carrier-free
Fibroblast Growth Factors
Antibodies:
FGF2
All Stem Cell Reagents