Three key points about biofilm models: 1) There are several in vitro and in vivo models for studying multispecies bacterial biofilms, each with strengths and weaknesses. 2) 3D printed biofilm models provide a robust and reproducible model for testing antimicrobials both in vitro and in vivo with less variability than other models. 3) When testing new antimicrobial therapies, researchers should use more than one biofilm model to validate results given the pitfalls of individual models.

1. © 2015 CMC Consulting Group. All Rights Reserved.
NEW 3D PRINTED BIOFILM MODELS FOR STUDYING
MULTISPECIES BACTERIAL COMMUNITIES
Mitch Sanders, PHD and Lindsay Poland, MS
Drug & Device Discovery Lab
Your Global Partner for Growth in Healthcare

2. 2
INTRODUCTION
Unlike free-floating planktonic bacteria that are quite
resistant to antibiotics and antimicrobials (such as
chlorhexidine (CHG) and nano crystalline silver), biofilms
are polymicrobial bacterial communities that are more
resistant to mechanical shear, antibiotics, and
antimicrobials.
Biofilms consist of a close network of bacteria that are
tethered together with a slime-like matrix mostly
consisting of exopolysaccharides, proteins, and nucleic acids (referred to as the EPS). This
dense community of bacteria has multiple layers with the top layer shedding active
planktonic-like bacteria while the deeper layers are more senescent (no longer capable of
dividing but still alive, from Latin: senescere, meaning "to grow old”).
3D BIOFILM MODELS
The challenge with testing antimicrobials with planktonic bacteria is that most culture
models do not reflect the complex molecular determinants that mediate quorum
sensing, sporulation, and other adaptive phenotypes that are representative of a true
biofilm. Several labs have made great strides in creating biofilm models using
bioreactors and cartridge-like drip models. In our experience, these models fail to set up
robust biofilms that are as durable as those developed in vivo. Imagine just for a
moment the biofilms and plaque that build up on our teeth while sleeping overnight or
the biofilm that grows on your pet’s water dish in less than 24 hours when you forget to
change the dish. Another example is the biofilm in a chronic wound that is resistant to
most antibiotics and antimicrobials including bleach solution.
In 2013, Connell and colleagues at the University of Texas demonstrated that they could
use a 3D printer to study bacterial communities. We have used this approach at 3DL
with an experimental 3D printer to establish polymicrobial biofilms that are more robust
and reproducible that can be tested both in vitro and in vivo in a modified mouse model.
The 3D printed biofilm models are much more consistent in terms of the amount of
protein and bacteria dispensed that can provide for more uniform replicates that have
less standard deviation of error than those established from the other well-
characterized biofilm models described below.
KEY POINTS
 Every biofilm model
has it pitfalls and
strengths.
 Use at least two
models to validate
the efficacy of your
antimicrobial
therapy.
 3DL can provide
robust biofilm
models to accelerate
your pre-clinical
development.
Figure 1 Atomic Force Microscopy (AFM):
This is an image of a bacterial biofilm of
Staphylococcus aureus, Pseudomonas
aeruginosa, and Streptococcus pyogenes
Figure 2 This 3D Printer is used in
printing complex bacterial
communities. A separate white paper
will be submitted to Wound Repair and
Regeneration demonstrating the
validity of the model. We use a
Makerbot 2x replicator configured
with a high precision (NE 1000) syringe
pump configured with a thermo-kinetic
heat clamp to form bacterial biofilms.

3. 3
CDC BIOREACTOR MODEL
The CDC bioreactor model is a well-established continuous flow model
for forming multi-colony biofilms that was developed by Donlan et al.,
(2004) in the CDC Biofilm Lab. This model is well suited for microscopy
because the coupon material can be punched out of the slides so that
several replicates can be obtained for each sample condition. A typical
reactor has multiple polypropylene coupon holders suspended from
the support lid. Liquid growth media/biocide/etc. is circulated through
the chamber, while the liquid is mixed by a magnetic stir bar to
generate mechanical shear. More recent studies indicate that
coupons made of polyetheretherketone (PEEK) material can set up
more durable bio-films (Williams et al., 2011).
STIRRED CELLS AND DRIP CELLS
Stirred and drip cells are other examples of continuous biofilm models that
were developed to account for the mechanical shear forces that drive the
formation of more stable biofilms developed at Montana State University,
(MSU, Bozeman, MT: Herigstad et al., 2001). The stirred cells have the
benefit of a well-established EPS matrix and biofilm while removing the
preponderance of planktonic bacteria. The Center for Biofilm Engineering
(CBE) at MSU continues to be one of the leading institutes in studying
biofilm models. CBE hosts a variety of symposia and workshops on
biofilms both for industry and academia alike.
SKIN EXPLANT MODELS
UV sterilized porcine skin was developed as a biofilm model by Greg Schultz’s lab demonstrating
that a saline rinse retained 109
bacteria but hydro debridement reduced the bio-burden to 104
CFU/g (Yang et al., 2013). The explant model has shown to be a useful model to set up biofilms
in under 7 days for P. aeruginosa but > 7 days for S. aureus. The porcine skin is a reasonable
surrogate for the mouse model described below given that the data is comparable to the mouse
model but less expensive. Critics of this porcine skin model
suggest that it does not reflect human skin and there is no
immune response. However, Schultz and colleagues have
demonstrated that this model can produce biofilms that
are quite robust and even resistant to treatment with high
concentrations of antimicrobials and even chlorine bleach.
Figure 3 CDC Type Bioreactor: This
continuous flow vessel has room for 6
channels that can be processed
simultaneously and can be used with a
plethora of material types, including
plastics, metals, and ceramics.
Figure 4 A stirred cell bioreactor
allows for the formation of robust
biofilms. This model system has
the ability to run multiple coupons
ille tempore. Other variations
include a rotating disk that uses
centrifugal shear forces to set up
robust biofilms.
Figure 5 Porcine skin is ideal for establishing host
pathogen binding studies, less variable than the in
vivid model.

4. 4
Biofilm Models
Figure 6 Contact Mitchell.sanders@cmc-co.net for pricing inquiries on Biofilm Models.
MOUSE MODELS
Mouse biofilm models allow researchers to study how
biofilms can stall wound healing in normal and diabetic
animals (Zao et al., 2010). However, if you are not
studying wound healing and only studying biofilm
formation, the in vitro models are probably more than
sufficient because they have less variability than the
mouse model system. Because of the inherent variability of
the mouse model it takes 9 mice per group to get statistical
significance. Many of our colleagues feel that this mouse
model is less favorable than the in vitro models because of this variability. Our hypothesis is
that 3D printed biofilms will make the mouse model more robust and more applicable by
reducing the variability and therefore the number of replicates required for this model. We plan
to present these new results at the next Symposium on Advanced Wound Care (SAWC) in the
Fall of 2015.
SUMMARY
There are several models to study biofilms. However, each model has its own pitfalls and strengths. We
recommend that researchers use more than one model to validate their testing protocol with the
antibacterial or antimicrobial combination product. When you think about which lab you should use for
biofilm studies, consider a lab that has at least 20+ years of experience in studying biofilms and
determine if they are capable of generating timely, statistically significant, and high publication quality
data.
Figure 7 The balb/c mouse is commonly used
in biofilm studies. This model is
far less expensive than the partial thickness
porcine infection model or the rabbit urinary
catheter model (not shown), but is more
variable.
Biofilm Models Multispecies Advantages Pitfalls Measurments
CDC Bioreacter ++ Moderate Throughput STD Model/Cumbersome CFU Plating
Rotary Disk ++ Measures Shear Force Cumbersome CFU Plating
Drip Module ++ Robust Biofilms Old Model/Cumbersome CFU Plating/Fl Confocal Microscopy
New 3D Bioprinting +++ Versatile for in vitro & in vivo models New System CFU Plating/Fl Confocal Microscopy
Porcine Skin + Direct interaction with host protiens No Host Response, oversimplified CFU Plating/Fl Confocal Microscopy
Mouse Chronic Infection ++ Closest to Chronic Wound Infection Higher error requires 9 replicates CFU Plating

5. 5
AUTHORS
Mitchell Sanders MS, PhD, is the Managing Director of the Drug and Device Discovery
Lab at CMC Consulting. Mitch has 30+ years of experience in studying bacterial biofilms
and chronic wound infections. With ECI Biotech, Mitchell has produced over 12 peer-
reviewed publications and 24 worldwide patents in medical device and in vitro
diagnostics. Mitchell is an expert in clinical and translational research and is a reviewer
for the Wound Healing Society, CIMIT, MassVentures, MIT, WPI, Tech Sandbox, Piranha
Pond, SBANE and the Venture Forum. Mitchell has an MS and PhD from WPI in
molecular biology and biomedical sciences with 2 Postdocs (biochemistry and pathogen
genetics) at the Whitehead Institute/MIT.
Lindsay Poland is a scientist at 3DL who has 10+ years of experience in studying clinical
microbiology and protein biochemistry. Lindsay is an expert in molecular biology and
protein biochemistry of chronic wounds. She has 14 years of experience with almost 11
of them being in the industry with Mitch Sanders at ECI Biotech (Worcester MA)
studying wound repair and regeneration and chronic wound infection.

6. 6
REFERENCES
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PMID:25959709
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475X.2010.00608.x. Epub 2010 Aug 19.

7. 7
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