Table 2.

Representative examples for recent^ain vitro biofilm models as of 2015

Graphic depiction	Description	Application	Microorganism(s) evaluated	Advantages	Limitations
Recent static in vitro biofilm models
3D bioprinted biofilm construct (Ning et al., 2019)
	Double crosslinked alginate-based bioink is used to print solid and porous 3D bacterial structures	Formation of mature biofilms for antimicrobial testing	E. coli clinical isolate (American Type Culture Collection [ATCC] 25922), methicillin-resistant S. aureus (MRSA, clinical isolate, ATCC 700788), methicillin-sensitive S. aureus (MSSA, clinical isolate, ATCC 29213) P. aeruginosa, PAO1 (wild-type strain, ATCC 47085) Cell density: Initial: OD600nm = 1.0 Final: Visually reported (3D reconstructed CLSM z stack images)	•Customizable predesigned biofilm thickness and dimensions •Construct stability for up to 4 weeks •Adequate cell viability •Suitable for aerobic and anaerobic biofilms •Allows biofilm formation monitoring •Suitable for antimicrobial testing (biofilm penetration and eradication) •Greater antimicrobial resistance than 2D cultures •Cost effective	•Constructs with thickness of 0.5 mm or thinner are structurally unstable •Missing interaction with cellular and molecular components •Decreased cell density as thickness increases •Restricted to biofilms grown in semisolid conditions
Patterned SLIPS (“slippery” lubricant-infused porous surface) (Bruchmann et al., 2017)
	Porous polymer areas with specific geometries are delimited by a lubricant that forms SLIPS regions. Biofilm formation occurs at the hydrophilic porous areas. The result is a series of 3D biofilms with uniform shapes and dimensions along the array	Biofilm screening Antimicrobial testing	P. aeruginosa strains PA01, PA30, and PA49 Cell density: Initial: 1 × 108 bacteria/mL Final: Not reported	•Control over the biofilm microcluster geometry •High reproducibility •Biofilm stability •Allows the study of interactions between biofilm subpopulations •Suitable for drug screening	•Biofilm formation by different bacterial strains may be influenced by the size and arrangement of SLIPS microclusters •Interactions between biofilm clusters •Missing interaction with cellular and molecular components
The dissolvable bead (Dall et al., 2017)
	Sodium alginate beads are incubated in liquid bacterial culture for biofilm formation. The beads are dissolved, and biofilms are released	Antimicrobial testing	MSSA S. aureus (ATCC #29213), S. mutans (NCTC #10923), E. coli (ATCC #25922), clinical isolates of coagulase-negative Staphylococcus (CNS-J), E. faecalis, K. pneumoniae, and P. aeruginosa Cell density: Initial: 104 CFU/mL Final: Mean cell number (CFU/mL) Log107.00 ± 0.39	•Localized biofilm formation onto the alginate bead surface •Uniform exposure to antimicrobials •Undisrupted biofilm •Suitable for drug screening •Greater assay responsiveness than the crystal violet assay to an antibiotic challenge •Rapid and reproducible •Cost efficient and time efficient	•Formed beads are not regular in size and shape •Potential influence of alginate beads architecture on biofilm phenotype •Homogeneous biofilm composition •Missing interaction with cellular and molecular components
Impedance-based multielectrode array (Goikoetxea et al., 2018)
	Biofilm is grown onto an MEA biosensor placed into a chamber to measure impedance changes at different frequencies, which are correlated to biofilm structural changes during its development	Structural characterization of bacterial biofilms	E. coli TG1, isogenic ΔcsgD, ΔcsgB, and ΔbcsA mutant strains Cell density: Initial: 2.8 × 108 cells/mL Final (cells/mL): from 2.8 × 107 to 4.2 × 107	•Nondestructive and label-free characterization of biofilms •Allows distinction of biofilms with structural differences •Able to identify attachment and maturation stages of the biofilm formation cycle •Good spatial resolution •Reduced sample volume	•Unable to correlate the obtained impedance readings at certain frequencies with metabolic activity or number of cells present •Missing interaction with cellular and molecular components
Vertical capacitance aptamer-functionalized sensor (Song et al., 2019)
	Biosensor functionalized with bacteria-aptamers, which are vertically connected to a measurement system during biofilm formation. Biofilm electrical properties are recorded	Bacterial detection and biofilm formation monitoring in blood	E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), S. aureus (ATCC 29213), mutant strains Δpel and Δpel/Δpsl of P. aeruginosa strain PAO1 Cell density: Initial: 100, 101, 102, or 103 CFU/mL Final: Not reported	•Real-time monitoring of biofilm formation •Biofilm formation and bacterial growth can be monitored simultaneously •Suitable for antimicrobial testing •Simple, flexible, and cost effective •Detection of low bacterial concentrations	•Potential influence of the aptamer-bacteria interactions on biofilm development •Missing influence of shear forces during blood flow
Gold mushroom-like nanoplasmonic biochip (Funari et al., 2018)
	Localized surface plasmon resonance (LSPR) biochip consisting of a detector with mushroom-like nanostructures where the biofilm is formed and monitored	Biofilm characterization and drug screening	E. coli (MC4100). Cell density: Initial: 2 × 107 CFU/mL Final: Not reported	•Real-time, nondisruptive, label-free monitoring of biofilm formation •High sensitivity •Allows comparisons between biofilm-forming species •Suitable for antimicrobial testing •Possibility of automatization for data acquisition during several days	•Requirement of specialized equipment for device fabrication •Potential scattering in the LSPR signal if there is a high cell density per well •Sustained illumination may cause local stress to cells •Intermediate viability (65%) •Missing interaction with cellular and molecular components
Biofilm rheometer plate (Grumbein et al., 2016)
	Biofilm is formed onto an agar surface contained along the two parts of a sample holder. A stretching force is applied to both sides	Quantification of mechanical properties (rupture forces and tensile strengths) in situ	B. subtilis B-1 Cell density: Initial: Not reported (15 μL of overnight bacterial liquid culture) Final: Not reported	•Biofilm can be measured in its naturally grown state •Allows the evaluation of antimicrobials on biofilm structural resistance •Cost effective: reusable sample holders •Biofilm deformation during the test can be followed visually	•Low force signal detection •Proper methods and filters must be applied to optimize the signal-to-noise ratio of measured data •Does not allow multiple biomechanical quantifications on the same sample •Missing interaction with cellular and molecular components
Human plasma biofilm model (hpBIOM) (Besser et al., 2020; Besser et al., 2019)
	Ellipsoid-like clot discs composed of a fibrin matrix from human plasma preserves and buffy coats plus incorporated biofilm-forming bacteria	Biofilm-infected human wounds	S. aureus subsp. aureus (DSM 799), S. epidermidis (DSM 20044), P. aeruginosa (DSM 939), E. faecium (DSM 2146), and C. albicans (DSM 1386) Cell density: Initial: 2 × 106 CFU/3 mL plasma solution Final (24 h): From 105 to 109	•Resembles a human wound milieu •Incorporates immune system components: white cells, platelets, and complement system •Personalized antimicrobial testing (donor specific)	•Results are not generalizable (effect is donor specific) •Pathogen-dependent stability •Does not include the damaged skin component of the wound: lack of skin cells
Recent dynamic in vitro biofilm models
Microcalorimetry flow system (Said et al., 2015)
	A heat exchange unit connects an external bioreactor to the stainless-steel ampoule of a commercially available calorimeter. The heat flow, which is associated with bacterial metabolic activity, is then related to changes in the power signal of the calorimeter as an indicator of biofilm formation	Antimicrobial testing and study of biofilm formation on different medical tubing materials	S. aureus (NCIMB 9518) Cell density: Initial: 106 CFU Final: 3 × 1010 CFU/mL	•Sensitive to reduced cell density and small changes in metabolic activity •Noninvasive & nondisruptive method •Suitable for real-time monitoring of biofilm formation •Simple and reusable (removable parts permit sterilization) •Versatile: Allows monitoring of bacterial activity and evaluation of the effect of antimicrobials in a wide range of tubing materials and medical devices/implants (does not require optical clarity of the sample)	•Limited to low shear rates so as to not disturb the calorimeter •Large sized equipment •High costs for the calorimeter equipment •Complex data analysis •Limited throughput •Nonspecific: Heat flow signal involves the sum of a variety of processes taking place •It would require important adaptations if medical devices/implants are intended to be evaluated: should consider cellular and molecular interactions
Duckworth Biofilm Device (Duckworth et al., 2018)
	3D-printed device with four channels that provide nutrients to three circular wells each. An agar disk, in direct contact with the nutritious flow, rests onto each well and supports a cellulose membrane, resembling the air-liquid interface where the biofilm is grown	Exuding chronic wound infection model for testing of antimicrobial wound dressings	P. aeruginosa S. aureus Cell density: Initial: 105 CFU Final: ∼8–10 Log(CFU/mL)	•Mimics the air-liquid interface found in wounds •Allows temperature control and visual inspection without disrupting the biofilm •Has a lid and filter to prevent samples from crosscontamination •Good reproducibility and versatility •Small sized, inexpensive, reusable, and simple setup	•Requires fairly large amounts of media (500 mL/24 h). •Level of exudate is not regulated •Missing interaction with cellular and molecular components
Flow chamber system for medical implants (Rath et al., 2017)
	A closed flow system where the flow chamber consists of different materials and spacers to create a specimen housing where easily interchangeable implant disc samples are placed	Testing of dental implant materials	(1)S. gordonii (DSM, 20568) (2)S. salivarius (DSM, 20067) (3)P. gingivalis (DSM, 20709) (4)S. oralis (ATCC 9811) (5)A.actinomycetemcomitans (ATCC 2474) Cell density: (CFU/mL, ×106) Initial: (1) 1.94; (2) 4.19; (3) 7.88; (4) 3.5; and (5) 1.25 Final: Not reported. Higher thickness: 38.85 μm for 7.88 × 106 CFU/mL	•Mimics physiological flow conditions of the oral cavity •Setup can be adjusted to aerobic or anaerobic conditions •Direct microscopic observation of the implant surface is possible •Easy removal of implant samples •Minimization of detachment effects because of removable cover and discs •Fully autoclavable and reusable system •Reproducible	•Culture conditions do not mimic the wide variety of nutrients found in the oral cavity •Optimization of conditions is necessary for the formation of multispecies biofilms •Missing interaction with cellular and molecular components
Flexible impedimetric detection platform (Huiszoon et al., 2019)
	A flexible platform of gold interdigitated electrodes (IDEs) is fitted into the lumen of a urinary catheter using a polyimide film as substrate. Bacterial growth medium is circulated through the catheter to allow biofilm formation, which is evaluated as changes in impedance	Real-time monitoring of biofilm development and evaluation of bioelectric effect as potential treatment	E. coli K-12 W3110 Cell density: Initial: 6 × 107 CFU/mL Final: Not reported. Impedance decrease of ∼30% after 24 h of biofilm growth	•Flexible nature of the platform allows its use in clinically relevant curved surfaces and potentially in other types of geometries •Compact size •Allows for real-time nondestructive monitoring of biofilm development •Suitable for evaluation of combined therapies/treatments	•Variability of impedance measurements limits sensitivity •Technically challenging •Missing interaction with cellular and molecular components
Microfluidic agarose channel device (Jung et al., 2015)
	The system consists of a glass slide-sized microfluidic chip with multiple channels where bacterial cells are embedded in an agarose matrix, generating biofilm-like structures (ECAS). Nutritious flow is irrigated onto the surface resulting in a shear-free environment	Study of surface-free biofilms, similar to those formed within mucosal or viscous environments Testing of antimicrobial agents	P. aeruginosa (ATCC 27853), E. coli (ATCC 25922), E. faecalis (ATCC 29212), S. aureus (ATCC 29213), B. subtilis (ATCC 6633), P. aeruginosa PA14 and its mutants wspF and pel Cell density: Initial: OD600nm ≈ 1.0 Final: Not reported	•Allows for direct microscopic investigation •Better mimics the environment found on shear-free clinically relevant intracellular- and mucosal-associated biofilms •Simple, small-sized, and inexpensive device •Full biofilm development cycle is reached in few hours (9–12 h)	•Model not relevant to environments under shear stress •Maturation and integrity of ECAS is affected by fixing stress, viscosity, and nutrition •Missing interaction with cellular and molecular components
Microfluidic wound model (Wright et al., 2015)
	A PDMS chip rests onto an agarose sheet with a central channel where a bacterial solution is added. The chip contains irrigation channels that contact and go along each side of the agarose central channel to create a concentration gradient	Study of wound biofilm structure and development Suitable for drug testing	P. aeruginosa (PAO1 & GFP-PAO1) E. coli (ATCC 700926) Cell density: Initial: 108 CFU Final: Not reported. Maximum biofilm surface coverage area ∼55%	•Small sized •Resembles concentration gradients of wounds •Viewing channels allow high-resolution imaging techniques for real-time monitoring •Allows identification of individual species in dual-species biofilms	•Limited throughput •Missing interaction with the cellular and molecular components found in wound biofilm microenvironment
Microfluidic artificial teeth device (Lam et al., 2016)
	A microfluidic device consisting of 8 × 16 parallel incubation chambers, on top of which a series of microstructures with particular functions are included to allow the control of different environmental factors during biofilm growth and development	Study of biofilm growth and development under adjustable environmental conditions pertaining to dental disease Suitable for testing of potential antimicrobials	Streptococci, F. nucleatum, A. naeslundii, P. gingivalis Cell density: Initial: 105–107 cells/mL Final: Not reported. Biofilm thickness of 100 μm	•Facilitates the real-time and long-term monitoring (≥1 weeks) of biofilm development •Ability to independently adjust several microenvironmental conditions in scheduled times •Allows quantitative determination of biofilm thickness, cell viability, and spatial distribution •Small-sized and inexpensive device	•Requires specialized technical abilities for device fabrication and experimental setup •Missing interaction with cellular and molecular components

^aThe models listed in this table include some examples of how different multidisciplinary research groups have proposed to approach and overcome certain limitations of previous and currently used biofilm models. Because of specific goals being pursued, equipment and/or methodologies proposed are mostly particular of certain laboratories and may not be easily implementable in most research facilities. Despite this, they provide out-of-the-box strategies to study and analyze biofilms and may serve as inspiration for the development of more accurate biofilm models.