1

Thermo-Responsive Polymers for Cell-based Therapeutic

Applications.

A thesis submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

MASTER OF SCIENCE

in the Biomedical Engineering Program

in the School Biomedical, Chemical, and Environmental Engineering

of the College of Engineering and Applied Science

November, 2014

by

Hodari-Sadiki James

B.A., Berea College, Berea, KY, 2012

Committee Chair: Daria A. Narmoneva, Ph.D.

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Abstract

Poly (N-isopropylacrylamide) (PNIPAAm) is a well-known thermo-responsive polymer that has be

shown to be biocompatible, with surfaces coated with PNIPAAm supporting the culture of cells. These

surfaces support the adhesion and proliferation of multiple cell phenotypes at 37 °C, when surface is

hydrophobic, as the polymer chains are collapse and lose their affinity for water. Reducing the

temperature below the polymers lower critical solution temperature (LCST) elicits hydration and

swelling of the polymer chains and leads to cell detachment. In vitro culture on thermo-responsive

surfaces can be used to produce cell sheets for the use of different therapeutic treatments. PNIPAAm

coated membranes were used to culture human keratinocyte cells to confluence, with cell release

possible after exposing the membranes to room temperature (~25 °C) for 10 minutes. Cell sheet

transfer was possible from the coated membrane to cell culture dishes using a protocol that we

developed. There was also a trend towards similar cell apoptosis on both PNIPAAm coated and

uncoated surfaces.

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4

Acknowledgements

I would like to acknowledge the University Of Cincinnati Department Of Biomedical

Engineering for Graduate Scholarship and Assistantship support which allowed me to fund my studies

at the University of Cincinnati. The research performed in this thesis was supported by funds from the

University of Cincinnati Department of Biomedical, Chemical and Environmental Engineering SEED

Grant.

I would like to express my gratitude to my advisor Dr. Daria Narmoneva for welcoming me into

her lab and giving me an opportunity to work on multiple biomedical research projects. Without her

unending support, guidance and encouragement, this process would have certainly been more difficult.

I am also indebted to Dr. Dale Schaefer, and Dr. T Douglas Mast, for taking the time to serve as

members of my thesis committee.

I would also like to thank Chukwuemeka Chikelu, Naiping Hu, Toloo Taghian, Andrea Lalley,

Brandon Brown, and Bo Yang for being valued colleagues and friends. I am also thankful to staff and

faculty members in Biomedical Engineering, particularly Dr. Jing-Huei Lee and Barbara Carter, for all

the help and support during my difficult times. Finally, I want to thank my mother, brother, and other

close family and friends for their love, and support and prayer throughout this entire process.

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Table of Contents

Abstract..............................................................................................................................2

Acknowledgements ............................................................................................................ 4

Table of Contents ................................................................................................................ 5

List of Tables and Figures .................................................................................................. 6

Chapter 1: Emerging Technologies for Cell Sheet Engineering using Thermo-

Responsive Polymers. ........................................................................................................ 8

Chapter 2 :Experimental Studies with Thermo-Responsive Membranes ........................ 21

2. 1 Background and Introduction: ................................................................................... 21

2.2 Cell Detachment and Transfer Study ......................................................................... 23

2.3 Comparison of Keratinocyte Apoptosis on Different Culture Surfaces ..................... 28

Chapter 3: Conclusions and Future Directions ................................................................ 32

Bibliography ..................................................................................................................... 34

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List of Tables and Figures

Chapter 1:

Figure 1: Schematic illustration of cell sheet on PNIPAAm modified surface. ................ 9

Table 1: Showing the Responses of Multiple Cell Phenotypes Cultured on Thermo-

Responsive Surfaces........................................................................................................ 10

Figure 1.2: An illustration of cell sheet detachment by different types of water supply . 13

Chapter 2 :

Figure 2.1: Wound healing schematic and the proposed treatment strategy .................... 22

Figure 2.2 Image of the plastic insert which contains the porous PVDF membrane that

was spin coated with PNIPAAm: ..................................................................................... 23

Figure 2.3 : Schematic showing the different surfaces that were explored in preliminary

studies.. ............................................................................................................................. 24

Figure 2.4 : Schematic of the cell detachment and transfer approach using collagen ..... 25

Figure 2.5 : 4x fluorescent image of KC cells after being transferred from PNIPAAm

coated membrane to sterile uncoated dishes………………………...………….………26

Figure 2.6 : 20x image of keratinocyte cells held in collagen after being transferred from

coated membranes to a culture dish. ................................................................................ 27

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Figure 2.7 : Schematic showing an overview of KC cell apoptosis study.. ..................... 28

Figure 2.8 (A): 20x image of KC nuclei co-stained with fluorescent DAPI (blue) and

caspase3 (red) ................................................................................................................... 30

Figure 2.8 (B) : 4x image KC nuclei co-stained with fluorescent DAPI (blue) and

caspase3 (red). .................................................................................................................. 30

Figure 2.9: Graph showing the percentage KC cell death………………………………… 31

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Chapter 1: Emerging Technologies for Cell Sheet Engineering using Thermo-

Responsive Polymers.

1.1 Introduction:

During a typical cell culture process, cell adhesion to the culture surface is important in order to

foster the healthy growth, differentiation, migration, and survival of cells [1], [2]. Adhesion, however,

presents a problem when cells grown in vitro need to be transported to an in vivo target area. The

conventional procedure involves proteolytic degradation of extracellular matrix (ECM) that holds the

cells together via enzymatic action, for example trypsinization. This procedure usually produces a

suspension of cells that can then be transported to another culture vessel. However, destruction of the

cell sheet's ECM is not very efficient from a tissue-engineering viewpoint, as the cell-cell interactions

present are usually integral to their in vivo function [3]. In addition nonspecific proteases can damage

crucial cell surface proteins [3], [4]. These major drawbacks have fueled research into alternative cell

harvesting methods.

Poly (N-isopropylacrylamide) (PNIPAAm) is a well-known thermo-responsive polymer which

has been studied since the late 1960's [5]. Its use as a culture surface for the in vitro growth of intact

cell sheets, however, was not studied until the early 1990's [4], [6]-[8]. In recent years, cell culture on

PNIPAAm coated surfaces or within PNIPAAm hydrogels, has been explored as a possible solution to

the problems presented by conventional cell harvesting methods [9]. A surface grafted with the polymer

has the unique property of switching from a hydrophobic to a hydrophilic state when the temperature is

decreased below its lower critical solution temperature (LCST) (~32 °C) [10]. A confirmation change

in the polymer chains of the grafted PNIPAAm layer, caused by reduced temperature, can elicit

detachment of confluent cell sheets as shown by the schematic in Figure 1 [4], [7], [11]–[16].

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This chapter will; 1) Review recent cell sheet engineering studies using PNIPAAm, or

PNIPAAm copolymers, as a cell culture surface, and 2) Come up with a unique approach of producing

keratinocyte cell sheets to be used in the treatment of chronic wounds. Focus will be placed on cell to

surface interactions such as, adhesion, proliferation, and detachment. Since cellular responses to

PNIPAAm grafted surfaces have been found to be both cell-type specific, and dependent on PNIPAAm

grafting thickness in previous studies [20],[25]. Promising applications of cell sheet engineering

research using PNIPAAm coated culture surfaces will also be highlighted at the end of this chapter.

Figure 1.1: Schematic illustration of cell sheet on PNIPAm modified surface. A: At 37 °C, cells can adhere on

dehydrated PNIPAm layer. B: At 20 °C, cells can detach from hydrated PNIPAm layer with ECM, resulting in

an intact and contiguous cell sheet [14].

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Surface Description

Cell Phenotype Cell Adhesion &

Proliferation

Cell Detachment Refs.

Flat (F) and Micro-patterned (MP)

PNIPAAm films on TCPS

Rat bone marrow

mesenchymal stem

cells (BMSC)

Static: ↑proliferation rate at 2 weeks

on both F and MP

surfaces.

Dynamic: ↑ Total cell proliferation at

21 days on MP surface.

N/A [17]

PIPAAm Graft on

Polystyrene Dish (PSD) &

Polystyrene Nano-fibrous Mat (PNM)

Human fibroblasts

↑ Adhesion and cell spreading on

PNM surface.

↑ Detachment on PNM compared

to PSD.

[13]

PNIPAAm on

Chitosan membranes

Human fetal lung

fibroblast cell line

(MRC-5)

Best proliferation rate on P-IPrOH75-

grafted surface

Complete cell sheet detachment on

P-IPrOH75 surface.

[18]

(PNIPAAm)-grafted on

Flat PDMS substrate &

Micro-patterned PDMS substrates

Bovine aortic vascular

smooth muscle

cell (VSMCs)

Good adhesion on flat surface &

micro-patterned surfaces with cell

alignment along channels.

Complete cell detachment on both

surfaces.

[19]

(PNIPAAm) on

Glass substrates

NIH/3T3 fibroblast

cells.

Best attachment and spreading on

graft thickness ≤ 14nm

Best detachment on thicknesses

between 5 and 14 nm

[20]

(P(NIPAAm-r-MPDSAH)) on

Glass substrates

NIH3T3 fibroblast

cells.

Very Good Attachment and

spreading on surfaces with ≤ 75:1

PNIPAAm:MPDSAH concentration

Complete detachment only on

surfaces with a with a 75:1

PNIPAAm:MPDSAH mixture.

[20]

PNIAM-co-AM on

TCPS

Human Chondrocyte

Cells

Very good attachment and spreading w/o PVDF 69% detachment [21]

PNIAM-co-AM on

TCPS

PNIPAAmSt microgel films

Spin-coated (SC) on glass &

Drop-coated (DC) on TCPS.

Human Chondrocyte

Cells

NIH/3T3 fibroblast

cells

Very good attachment and spreading

Attachment and spreading similar on

both uncoated and DC TCPS

with PVDF100% detachment [21] [22]

90% cell detachment in 2-3 mins

from DC TCPS with pipetting.

PNIPAAmSt microgel films

Spin-coated (SC) on glass &

Drop-coated (DC) on TCPS.

Gelatin/PNIPAAm-PHB-PNIPAAm

Copolymer on Glass cover-slips

(GCS)

NIH/3T3 fibroblast

cells

Mouse embryonic stem

(ES) cells

Attachment and spreading similar on

both uncoated and SC glass

substrates

↑ detachment on SC 1.0 wt %

microgel films compared to SC 0.5

and 0.75 wt % films

[22] [23]

↓ proliferation rate compared to

gelatin coated GCS

↑ cell detachment compared to

other surfaces

NGMA coated tissue culture plates

Corneal endothelial

cells (CEC)

grew to confluence

within seven days

4 °C the cell sheet

detached

[24]

Flat (F) and Micro-patterned (MP)

PNIPAAm films with elastin-like

protein (ELP) on TCPS

Rat bone marrow

mesenchymal stem

cells (BMSC)

Static: Proliferation similar on F and

MP to noELP surface.

Dynamic: ↑ Total proliferation on

both F & MP surfaces.

N/A [17]

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Surface Description

Cell Phenotype Cell Adhesion &

Proliferation

Cell Detachment Refs.

CD-90 immobilized-

P(IPAAm-co-CIPAAm) on TCPS

I) Thymic carcinoma

cell line (Ty-82).

II) Human dermal

fibroblasts (NHDFs).

III) Primary Mouse

Adipose tissue-derived

(ADP) cells

Best adhesion and spreading of Ty-82

cells and NHDFs on avidin-biotin

binding surfaces.

Ty-82 & ADP cell detachment

required application of shear stress

& low temperature treatment.

NHDFs detached from all coated

surface with low temperature

treatment.

[25]

bFGF-bound heparin-fictionalized

P(IPAAm-co-CIPAAm) on TCPS

NIH/3T3 fibroblast

cells.

Cells reached confluence in 3 days on

surfaces with bFGF

densities ≥ 20ng cm-2 compared to 5

days on TCPS dishes.

BFGF-8 and bFGF-20 showed best

detachment rate.

[26]

(PCL-g-P(NIPAAm)) films NIH/3T3 fibroblast

cells

↑ proliferation with increased

collagen content.

↓ Cell detachment with increase in

collagen.

[27]

Table 1: Responses of Multiple Cell Phenotypes Cultured on Thermo-responsive Surfaces.

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1.2 Cell interactions with PNIPAAm Coated Surfaces

Multiple studies have shown that cell adhesion and detachment are dependent upon the thickness

of the PNIPAAm layer that is grafted onto the underlying base surface [11], [12], [20], [28]. Grafted

layers thinner than 14 nm showed good cell adhesion in a study carried out by Kong et al. However, in

Kong's study, low-temperature detachment of cells from the PNIPAAm coated glass substrates was

only optimal on surfaces with 11 nm and 13 nm thicknesses respectively, cells on detached poorly from

layers less than 5nm thick [20]. Different concentrations of the polymer Poly ((3-

(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)- ammonium hydroxide) (PMPDSAH) were

added to PNIPAAm, creating new polymer composites to improve cell detachment [20]. They observed

complete detachment from surfaces grafted with 4 nm thickness of (P(NIPAAm-r-MPDSAH)) which

had a 75:1 PNIPAAm to MPDSAH composition [20].

The improvement of cell detachment has been approached in multiple ways. Thai researchers

observed improved cell release from a temperature-responsive poly (N-isopropylacrylamide)-co-

acrylamide (PNIAM-co-AM) surface with the help of a hydrophilically modified

poly(vinylidenefluoride) (PVDF) membrane [21]. The group cultured human chondracyte cells on

their co-polymer coated surface, but only observed 69 ± 11.45% cell release when dishes were

incubated at 10 °C for 30 minutes [21]. The use of the PVDF membranes along with low temperature

treatment, not only improved the cell yield to 100%, but also facilitated cell sheet transfer and

decreased cell sheet shrinkage [21].

So far we have only looked at culture on traditional coated base surfaces, such as TCPS and glass

substrates, which are known to support in vitro cell culture. There are two studies in Table 1 that

explore the use of non-traditional base surfaces coated with PNIPAAm to improve adhesion, growth

and detachment of specific cell phenotypes. In 2008 Da Silva et al. used PNIPAAm coated chitosan

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membranes as a thermo-responsive culture surface. The group correlated chitosan's biocompatibility,

non-toxicity and membrane porosity with improved mass transfer of nutrients and metabolic wastes

[18], [29]. Results from the study showed favorable adhesion and spreading on coated surfaces, with

ungrafted chitosan membranes showing very poor cell compatibility [18]. Plasma treatment of coated

surfaces increases not only the surface's cell proliferation, but also enhances the detachment of

confluent cell sheets after 16 °C incubation [18].

More recently, a Korean study, led by Hwan Hee Oh, fabricated thermo-responsive polystyrene

(PS) nano-fibrous mats, by the electro-spinning of PS solution. These mats were then grafted with

PIPAAm to produce thermo-responsive PS nano-fiberous mats for use in in vitro cell culture [13]. They

observed improved adhesion and growth of human fibroblasts that were cultured on coated PS mats as

opposed to coated PS dishes [13]. The investigators proposed that the three-dimensional structure of

electrospun mats provided better conditions for cell metabolism, which resulted in improved

proliferation. There was no cell detachment on uncoated surfaces. However cells detached from both

polymer coated PS mats and dishes, with a higher percentage of cell detachment seen on coated PS

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nano-fibrous mats [13]. The porous structure of the mats was identified as the reason for rapid

hydration and improved detachment compared to solid surfaces [13]. The schematic in Figure 1.2

illustrates how different hydration methods can cause variations in cell detachment.

The culturing of cell sheets with specific architectures is desirable since most normal tissue has

some structure that is difficult to replicate in vitro. The effect of surface chemistry, mechanical stress

and topography on bone marrow mesenchymal stem cells (BMCS) cultured on PNIPAAm -coated

surfaces was investigated by Ozturk et al. Micro-patterned PNIPAAm films encouraged cell nuclei

alignment and elongation parallel to the direction of trenches fabricated on the surface [17].

Unpatterned surfaces exhibited random growth [17]. Detachment studies were not performed because

the goal was to impose mechanical stress on the cells via temperature variation [17].

Williams et al. attempted induced cell alignment by culturing vascular smooth muscle cells

(VSMS) on a thermo-responsive surface micro-patterned with the cell adhesive protein fibronectin. The

group maintained alignment within 30° of the fibronectin lanes [30] with successful detachment and

transfer of the aligned cell sheets [30]. This group also observed good adhesion and growth of VSMCs

on PNIPAAm-grafted micro-textured PDMS surfaces. Hoechst staining revealed elongated nuclei

aligned with lanes of the textured surface [19]. Complete cell sheets detached after low temperature

exposure, with reduced shrinkage compared to sheets grown on flat PNIPAAm-coated PDMS surfaces

[19].

The majority of the studies employed the photo polymerization method. This method allows for

chemical bonding between the grafted polymer layer and the underlying base surface, and is believed

foster better cell response and detachment [31], [32]. Thermo-responsive p(NIPAm-co-St) was used to

spin-coat glass substrates and drop-coat TCPS dishes in Xia et al's study. Styrene was included in the

co-polymer to optimize the amphiphilicity of the microgels in an attempt to improve cell detachment

[22]. NIH/3T3 cells showed similar cell adhesion and spreading properties on both spin-coated and

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uncoated glass substrates. However there was no cell detachment observed upon low temperature

treatment (20 °C) with cells only becoming more rounded, and requiring the application of shear stress

to be removed from the surface [22]. Drop coated surfaces only supported good adhesion and spreading

with a 0.5 wt % polymer composite coatings [22]. The investigators again reported the need for shear

stress along with low temperature to detach cells [22].

Loh et al. also looked at drop coating method for creating a thermo-responsive cell culture

surfaces. Loh's team used the tri-block copolymer, poly(N-isopropylacrylamide)- block-poly[(R)-3-

hydroxybutyrate]-block-poly(N-isopropylacrylamide) (PNIPAAm-PHB-PNI- PAAm), to drop coat

glass coverslips, and then added a layer of gelatin onto the polymer surface [23]. Mouse embryonic

stem (ES) cells were observed to proliferate better on the gelatin-triblock polymer coated surface, than

on surfaces coated with gelatin/PNIPAAm [23]. Detachment was tested by incubating substrates at 4 °C

for 20 minutes, with 91.3% ± 17.6% detaching from gelatin/triblock polymer, compared to 59.2% ±

10.4% from gelatin/PNIPAAm coated surface [23].

Most cell culture protocols employ growth factors, which are soluble stimulants used to

accelerate proliferation [33]. Because growth factors induce the degradation of other signaling

molecules surface immobilization of growth factors is seen as a promising method to reduce

degradation [33], [34]. A Japanese group explored thermo-responsive culture surfaces using poly(N-

isopropylacrylamide-co-2-carboxyisopropylacrylamide) P(IPAAm-co-CIPAAm) [25], [26]. They

covalently tethered heparin onto the CIPPAAm chains, and then added heparin binding proteins, such

as CD-90 and basic fibroblast growth factor (bFGF) to the surface [25], [26].

In the first study, CD-90 antibody-immobilized P(IPAAm-co-CIPAAm) TCPS surfaces were used

to culture two cell lines, thymic carcinoma cell line (Ty-82), and neonatal normal human dermal

fibroblasts (NHDFs), both of which are known to express CD-90 on their cell surface [25]. TY-82 cells

adhered better than NHDFs to the antibody-immobilized surfaces. However both cell types formed a

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confluent cell sheet after 4 days. There was only partial detachment of TY-82 cells after low

temperature treatment (20°C) on all grafted surfaces, with the application of shear stress required to get

complete detachment [25]. NHDFs spontaneously detached in response to the same low temperature

treatment, indicating that the detachment process is “cell specific” [25].

The same group looked at cell responses to surface immobilization of the growth factor bFGF on

the thermo-responsive surface [26]. NIH/3T3 fibroblast cells were cultured on bFGF-bound heparin-

fictionalized P(IPAAm-co-CIPAAm) surfaces with varying densities of bFGF. Cells reached confluence

in 3 days on surfaces with bFGF densities of 20 ng cm-2 and above, two days faster than cells cultured

on non-immobilized surfaces [26]. Density of bFGF also affected cell detachment with cells taking 45

minutes to detach from surfaces with densities of 8 and 20 ng cm-2 , compared with 120 minutes for

densities above 100 ng cm-2 , with the fastest detachment, 30 minutes, seen on non-immobilized

P(IPAAm-co-CIPAAm) surface [26]. The benefit of better adhesion and faster culture time on certain

bFGF-bound heparin-fictionalized P(IPAAm-co-CIPAAm) surfaces is helpful despite the drawback of

decreased detachment rate [26].

Table 1's final entry is a study of thermo-responsive poly (N-isopropylacrylamide)-grafted

polycaprolactone (PCL-g-P(NIPAAm)) films that were immobilized with cell-adhesive collagen to

improve cell sheet formation [27]. The higher the content of the immobilized collagen, the higher the

density of the attached cells [27]. PCL-g-P(NIPAAm)-collagen with thinner collagen coatings had

100% detachment. The percentage detachment decreased with increased collagen thickness [27].

1.3 Applications of Cell Sheet Engineering

Given the promising results on growth and release of different phenotypes on PNIPAAm

substrates or hydrogels it is logical that different research groups have looked into the medical

applications of cell sheet engineering.

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One such application is the construction of mesenchymal stem cell (MSCs) sheets for use in

tissue regeneration without the differentiation of the stem cells. MSCs have self-renewal ability and

multi-differentiation potential, making them ideal for treatment of certain debilitating injuries and

diseases [35]. In Yang et al's study, primary rat bone marrow-MSC (BM-MSCs) and human adipose

tissue-MSC (AT-MSCs) were cultured on PNIPAAm copolymer films in an attempt to reduce the loss

of its cell adhesion and differentiation caused enzymatic treatment [36]–[38]. Although the mechanism

by which MSCs lose their primitive nature is not fully understood, the deficiency in their plasticity may

arises from multiple exposure to proteolytic enzymes that degrade extracellular matrix (ECM) proteins

[39], [40]. Using temperature release instead of trypsinization should prolong the primitive nature

during in vitro culture [35].

Yang's group cultured intact cell sheets on PNIPAAm copolymer surfaces [35]. They observed

greater cell viability, more colonies and stronger differentiation of MSCs harvested via temperature

drop in comparison with the trypsinization [35]. This result was linked to the preservation of ECM

proteins caused by whole sheet retrieval using the thermo-responsive surface [35]. In Ozturk et al's

study demonstrated that rat BM MSCs retain their primitive nature after in vitro culture. Here MSCs

cultured dynamically on a PNIPAAm surface showed reduced MSC differentiation when compared to

static culture [17].

In contrast to Yang’s work, Peroglio et al sought to foster the differentiation of MSC cells in vitro

for treatment of intervertebral disc (IVD) pathologies. Injection of human MSC (hMSCs) into the

intervertebral disc (IVD) space is being explored to combat IVD degeneration [41]–[43]. A drawback

to this treatment is the harsh conditions present within the disc tissue. Low glucose, oxygen levels, pH

adversely impact undifferentiated MSCs [44]. The quality of repair hinges on the ability of the MSCs to

differentiate to the disc cell phenotype after injection, and also on the cell carrier that is used to initially

support the injected cells. Pre-culture of hMSCs to encourage differentiation toward the IVD cell

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phenotype before injection is a possible path to improved outcome [46]. Peroglio's ex vivo study, used

the thermo-reversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-

PNIPAAm) for the pre-culture of hMSCs, and also as a carrier for cell delivery. The hydrogel's

reversible gelation caused by its PNIPAAm content, made it more suited for pre-culture of hMSCs

before delivery to the target area [47]. The HA-PNIPAAm hydrogel induced hMSC differentiation

toward the disc phenotype more effectively than alginate gel, with neither pre-differentiation nor

growth factor supplementation required for hMSC disc-like differentiation [47].

Schwann cell transplantation has emerged as an attractive candidate in treating demyelinating

diseases resulting from peripheral and central nervous system injuries [48]. Most of the existing

methods involve the in vitro culture of schwann cells, which are then harvested and transported to the

target site via direct injection or via scaffold seeding [48]. These conventional harvesting and transfer

methods often involve proteolytic treatment, which leads to the degradation of cell-surface proteins and

also breakdown of important cell-cell interactions [49]. Pesirikan et al explored the construction of

schwann cell sheets on polystyrene tissue plates coated by PNIPAAm, which preserves cell-surface

proteins and cell-cell interactions through temperature initiated release. The investigators were able to

construct sheets of viable schwann cells that maintained cell–cell interactions as indicated by high

expression of cadherins as well as the level of neurotrophins [48].

Cell sheet engineering has also been used clinically in the treatment of injured corneas caused by

trauma or disease [50]. Japanese scientists constructed epithelial cell sheets by culturing autologous

oral mucosal epithelial cells on a thermo-responsive surface, which included the PNIPAAm polymer

[50]. Although the sample size of this study was small (4), the group reported a 100% success with

complete reepithalization of the previously damaged corneal surface in less than a week after the cell

sheets were introduced [50].

In a more recent study, human undifferentiated immature dental pulp stem cells (hIDPSC) were

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used to construct cell sheets for implantation on the cornea of rabbits with limbal stem cell deficiency

(LSCD). LSCD can be caused by multiple conditions, including genetic disorders, contact lens–

induced keratopathy, and iatrogenic multiple ocular surgeries [51]. The pathology causes a persistent

corneal epithelial defect or abnormal re-epithelialization by conjunctival epithelial cells, and can be

treated by a transplant of tissue from the patients’ non-affected eye [52]–[54]. Gomes et al looked at

culturing hIDPSCs cell sheets in vitro on a thermo-responsive surface, and transplanting the sheets onto

the cornea of impaired rabbits. Results showed continued improvement in corneal transparency in

comparison to the control group that had total opacification [51].

The final application considered is repair and regeneration of skin defects in mice. Cerqueira et

al. engineered a construct made by stacking multiple layers of human keratinocytes (hKC) cell sheets,

dermal fibroblasts (hDFb) cell sheets, and dermal microvascular endothelial (hDMEC) cell sheets,

which were cultured in vitro on thermo-responsive culture dishes [55]. Recognizing the complexity of

skin regeneration, the investigators choose hKCs for their wound re-epithelialization, hDFbs for ECM

remodeling, and hDMECs for dermal angiogenic signaling [56]–[58]. The rate of wound closure

improved in wounds treated with the cell-sheet constructs [55]. The outcome was dependent on the

arrangement of the different cell types in the construct, with some stacking configurations showing

only minimal wound healing improvement when compared to the control [55].

1.4 Proposal for a Novel Thermo-Responsive Cell Culture Surface

The information gathered from this review of cell interactions with these thermo-responsive

surfaces allowed us to construct our own approach for the culture, retrieval, and transfer of KC cell

sheets. Spin coating was chosen as our method of grafting PNIPAAm to the base surface due to its

shorter processing time and ability to support cell growth based on previous studies [22]. To counter act

the issues that were observed with cell detachment from spin coated surfaces however we proposed the

20

use of a porous PVDF membrane as our base surface.

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Chapter 2: Experimental Studies with Thermo-Responsive Membranes

2. 1 Background and Introduction:

Tissue engineering is a promising field of medicine which aims to develop biological substitutes

that restore tissue functions [59]. Wound closure is a key process during natural wound healing, as the

presence of barrier between the affected tissue and outside elements not only reduces the risk of

infection, but also creates a stable micro-environment for the regeneration of the extracellular matrix

(ECM) by cells [60]. The natural wound closure process is hampered in diabetic patients due to a

dysfunctional ECM and deficient blood vessel formation process [61], [62]. A common treatment of

diabetic wounds is wound management, which involves the regular cleaning and coverage of the

affected area to assist the natural healing process [63].

In the past our group has looked at the novel use of a nanofiber hydrogel as an ECM scaffold to

improve healing in the diabetic wound micro-environment [61], [62]. More recently, we have focused

on providing protection for unstable region while the healing process is taking place. To do so we have

proposed the in vitro growth of a keratinocyte cell sheet to be used in wound coverage, and is

illustrated in Fig 2.1 shown below. As explained in Chapter 1, cell sheet recovery using traditional cell

culture methods (eg. enzymatic action) is nearly impossible, as it requires the disassociation of the

cultured cell layer into a solution during the harvesting process [3]. The growth of KCs on a thermo-

responsive surface is a novel approach to produce a transferable cell sheets for use in diabetic wound

treatment.

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In this study we focused on the development of a method to culture and harvest human KCs on

thermo-responsive PNIPAAm coated PVDF membranes. In the first section of the study, we

investigated different cell detachment and transfer techniques, using information gained from extensive

literature review, as described in Chapter 1. The second section of our study focused on demonstrating

that PNIPAAm coating does not negatively affect cell viability, by comparing the levels of KC

apoptosis (programmed cell death) between different PNIPAAm substrates and plastic control.

Figure 2.1: Wound healing schematic (left) and the proposed treatment strategy (right). The normal wound

shows the presence of growth factors (GFs) and extracellular matrix (ECM) laid down by cells as well as

formation of blood vessels to the wound site. In contrast, in diabetes, there is increased inflammation and cell

death, degradation of GFs and ECM, leading to non-healing diabetic ulcers. The proposed treatment (right)

includes filling the wound with the stable nano-fiber matrix for cell infiltration, covered by keratinocyte sheet

layer delivered by smart release technology.

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2.2 Cell Detachment and Transfer Study

2.2a Overview

In this first section, porous PVDF membranes housed in plastic inserts, shown in figure 2.2, were

spin coated with PNIPAAm before they were used to culture KCs. The ability of these coated

membranes to support cell adhesion and proliferation were observed qualitatively. The objectives of

this study were to; 1) Conduct a preliminary study to determine optimal PNIPAAm coating parameters

for cell attachment and growth; 2) Demonstrate cell sheet detachment from the PNIPAAm coated

membrane, and 3) develop a procedure for the transfer cultured sheets to a secondary in vitro site.

2.2b Methods

Cell Culture

Frozen human primary Keratinocytes (KCs) obtained from healthy skin cell donors at the University of

Cincinnati Hospital were thawed and cultured in 75cm2 (T-75) cell culture flasks (Fisher brand) in

Medium 154 (Life Technologies, Grand Island, NY), supplemented with 0.1% CaCl2 0.2mM (Life

Technologies, Grand Island, NY), 1% antibiotic/antimycotic (Atlanta Biologicals, Lawrenceville, GA)

Figure 2.2 Image of the plastic insert which contains the porous PVDF membrane that was spin coated

with PNIPAAm. The thin fabric-like membrane is surrounded by a circular plastic barrier, which forms

an inner well around the membrane.

24

and Human Keratinocyte Growth Supplement (Life Technologies, Grand Island, NY). Cell cultures

were maintained at 37°C in 100% humidified air containing 6% CO2. Cells of passage 1-3 were used in

all experiments, in order to maintain the phenotype of the KCs.

Determination of Coating Parameters that Supported Cell Attachment and Growth.

To determine the best PNIPAAm casting parameters for PVDF membranes to support KC cell growth,

membranes were spun and drop coated with three different concentrations (0.5 % wt, 1.0 % wt, and

2.0 % wt) of PNIPAAm. The schematic in figure 2.3 shows an overview of this preliminary study.

Cell Culture on Thermo-Responsive Membranes

From culturing of KCs on the different PNIPAAm coated membranes described above, we observed

that cells attached and proliferated best on surfaces spin-coated with 2%wt PNIPAAm. Therefore, 2-

wt % PNIPAAm was used to spin-coat the membranes held within the inserts (30mm diameter, 0.4µm

pore size, 4.2 cm2 effective membrane; Millipore, Billerica, MA), followed by pretreatment with

formaldehyde. All coating procedures were carried out by our collaborator Dr Naiping Hu in Prof. Dale

Schaefer’s laboratory. Keratinocytes (KC) harvested from the T-75 culture flasks, via trypsinization,

and seeded on PNIPAAm coated membranes (n=2) that were sterilized with ethanol. The cells were

seeded at a density of 4.5×104 cells/cm2.

Figure 2.3: Schematic showing the

different surfaces that were explored in

preliminary studies to determine the best

surface to support KC cell growth.

25

Cell Sheet Detachment and Transfer

After 4 days, cells were stained with Orange Cell Tracker Dyes, CMTMR (Invitrogen, Carlsbad, CA).

Two approaches for cell detachment were attempted. The first involved the transfer of the membrane to

cell culture plates (Fisher Scientific, Pittsburg, PA) with cells inverted onto the surface of the dish.

These sections were then left at room temperature with a drop of media for 10 minutes to allow for cell

detachment, after which the membrane was peeled off the surface using sterile tweezers. In the second

approach, illustrated in Figure 2.4, cells were covered with 2.2 mg/ml collagen solution (Inamed

Biomaterials, Fremont, CA) mixture containing 5% 0.1M DPBS, 5% 0.2M HEPES and 55% PBS, and

incubated for 1 hour. The membrane from this insert was then transferred at room temperature to cell

culture plates with the collagen-cell layer inverted onto the surface of the well. After 10 minutes at

room temperature the membrane was peeled off using sterile tweezers. The detached cells in both

approaches were fixed with 2% formaldehyde, rinsed with PBS, and aqueous mounting medium

(Vector Laboratories, Burlingame, CA) was added afterwards. Images of detached cells were then taken

using an inverted fluorescent microscope (Olympus IX81; Olympus America, Center Valley, PA).

Figure 2.4: Schematic of the cell detachment and transfer approach using collage. Cells cultured on

PNIPAAm coated surface for 4 days before a layer of collagen is used to cover the cells. The

membrane, cell, and collagen structure is then inverted onto a cell culture dish at room temperature,

before the membrane is peeled off.

26

2.2c Results

The KC cells adhered and proliferated well on the 2% wt PNIPAAm coated membranes. After 4

days in culture a uniform layer of KCs were present, with cells bearing the same cell morphology as

those cultured in cell culture flasks. Fig 2.5 shows 4X images of KCs after being detached from the

PNIPAAm coated membranes using both transfer methods. The left image shows transfer cells from the

coated membranes directly, where cells were observed to detach in clumps. In contrast, cells transferred

after the addition of collagen layer showed more uniform cell-sheet detachment from the coated

membrane, shown in the right image.

Cell detachment from the coated membranes was observed after the membranes were left at room

temperature for very short time periods, between 5 and 10 minutes. The cell sheet could be viewed

floating in media without the use of a microscope, after detaching from underlying membrane surface.

This made it difficult to transfer cell sheets without the addition of collagen, as cells would be detached

from the membrane before inversion onto the cell culture dish. However with collagen added to the

layer of cells prior to membrane transfer, we observed detachment of cells on the well surface after the

Figure 2.5. (Right) Addition of collagen layer over cultured human keratinocytes on PNIPAAm substrate resulted in a better and more uniform sheet release, as compared to the more clump-like cell release without the collagen support (Left). Bar-150um.

27

membrane was peeled off. The difference in cell detachment using these two transfer methods is shown

in the 4x images in figure 2.5. Figure 2.6 shows a 20x image of cells after transfer using collagen, with

the cells maintaining a 3d structure in the collagen after transfer.

Figure 2.6: 20x image of keratinocyte cells (stained red) held in collagen (stained green) after being

transferred from coated membranes. The image shows that the cell/collagen construct was able to

maintain a 3D structure after transfer. Bar 5 𝜇m.

28

2.3 Comparison of Keratinocyte Apoptosis on Different Culture Surfaces

2.3a Overview and Objective

The objective of the study was to determine if the PNIPAAm coated surface does not negatively

affect KC cell viability. Based on our observations from the carrying out the cell detachment and

transfer study (Section 2.2) we hypothesized that there would be no detectable difference in KC

apoptosis between PNIPAAm coated surfaces (membranes and dishes) and uncoated surfaces. This

hypothesis was tested by measuring the percentage of caspase-3 positive KC present on PNIPAAm

coated membranes, PNIPAAm coated dishes and uncoated dishes after 24 and 72 hours of culture.

Figure 2.7 shows an overview of this study.

2.3b Methods

Cell Culture

Same as in Chapter 2.2b

Cell Culture on Thermo-Responsive Membranes

Same as in Chapter 2.2b except the cell seeding density for each membrane was changed to 105

cells/cm2 with one measurement (n = 1 ) for each time point (24 hours and 72 hours). One

measurement was viewed as sufficient since the area of a membrane was more than three times that of

Figure 2.7: Schematic showing an overview of KC cell apoptosis study. KC are seeded on three different

surfaces, PNIPAAm coated membranes, PNIPAAm coated dishes and uncoated dishes. Cells were then

cultured for 24 and 72 hours before staining with DAPI to identify all cell nuclei, and Caspase-3 to identify

apoptotic nuclei (red shaded cells).

29

the individual coated and uncoated wells.

Cell Apoptosis Detection

Three experimental groups were established: Cells seeded on (1) PNIPAAm-coated PVDF membranes

(n = 2), (2) a PNIPAAm-coated 24-well plate (n = 4 per time point) (UpCellTM Thermo Fisher

Scientific, Roskilde, Denmark), and (3) uncoated 24-well plate (n = 4 per time point) serving as the

control experiment, at a seeding density of (5 × 103 cells/cm2) for both 24 h and 3 days of culturing (n

= 4 per experimental group). At the end of the respective culture periods, cells cultured on all three

surfaces were fixed with 2% formaldehyde and immunostained afterwards with active caspase-3

monoclonal antibody (Life Technologies, Grand Island, NY) followed by goat anti-rabbit Alexa Fluor

488 antibody (Invitrogen, Eugene, OR) and DAPI.

Fluorescent Microscopy and Cell Counting

An inverted fluorescent microscope (Olympus IX81; Olympus America, Center Valley, PA) was used to

take images of the fixed cells on all three surfaces at 4X magnification. With DAPI staining

fluorescing at a different wavelength than Caspase-3 and allowing quantification of percentage cell

death. Figure 2.8A shows the result of the staining process at high magnification (20x), with the

nucleus of a dead cell colored both blue and red. Images were randomly taken at five different areas

around the well or membrane. The cells were then counted using Adobe Photoshop™ image software.

Figure 2.8B shows a 4X image randomly taken of a section of an uncoated dish, with all cell nuclei

stained blue and apoptotic cell nuclei also stained red.

Statistical Analysis

A univariate ANOVA with two factors – time (2 levels) and substrate (3 levels) was carried out on the

data set after the counting procedure was finished.

30

2.3c Results

During cell culture it was observed that the KCs attached and proliferated better in the uncoated

dishes than on the PNIPAAm coated dishes. Good cell growth was also observed in the coated inserts,

mimicking what was seen in the first section of the study, an indication that successful culturing of KC

on this surface is a repeatable process.

DAPI and Caspase-3 staining showed that percentage cell death after both 24 and 72 hours of

culture was greatest on PNIPAAm coated dishes. With Fig 2.6 illustrating 4.5 ± 0.4% and 5.5 ± 0.3%

cell apoptosis after day 1 and day 3 respectively on coated dishes. The figure also shows similar

percentages for apoptosis on uncoated dishes and coated membranes after one day of culture. After day

3 apoptosis was less on the coated membrane than both the uncoated and coated dish, with only 1.7 %

cell death. It is possible that here again, the porous nature of the membranes allowed for improved

nutrition and waste management between the KCs and the culture media, or better attachment of

PNIPAAm polymer to the surface.

Statistical analysis carried out on our data set showed that these results were not statistically

(B)

Figure 2.8: (A) 20x image of KC nuclei co-stained with fluorescent DAPI (blue) and caspase3 (red).

Nuclei that are stained with both blue and red indicate dead cells. Bar 5 um. (B) 4X image of a random

section of an uncoated dish, all cell nuclei present (Blue) from DAPI staining and all caspase-3 positive

cells (red) were counted to calculate the percentage cell apoptosis on each surface.

(A)

31

significant (two-way ANOVA testing for the effects of substrate and time in culture). Post-hoc

comparison with Bonferroni correction demonstrated a trend (p-value of 0.14) for the increased

apoptosis levels for PNIPAAm coated dishes (UpCellTM) compared to PNIPAAm coated membranes

and uncoated dishes, as also evidenced by the standard deviation bars in Figure 2.9

Figure 2.9: Culture of human keratinocytes (epithelial cells) on PNIPAAm-coated porous membrane

did not increase cell apoptosis (Caspase 3 marker), as compared to standard plastic dish control. In

contrast, there was a trend (p=0.14) for the increased apoptosis for keratinocytes cultures using

commercial UpCellTM PNIPAAm-coated dishes.

32

Chapter 3: Conclusions and Future Directions

From Chapter 1 we can clearly see that PNIPAAm grafted surfaces and hydrogels can be used for

the successful culture of multiple cell phenotypes. With specific cell responses such as adhesion,

growth, alignment, and differentiation being controlled via the composition, thickness, density and

topography of the PNIPAAm surface. Promising applications of cell sheet engineering in medical and

tissue engineering fields are also being explored given the biocompatibility of thermo-responsive

PNIPAAm surfaces and their ability produce viable cell sheets.

Our group’s exploratory studies into using KCs cultured on a thermo-responsive membrane as a

part of a treatment method for diabetic wounds are promising. As qualitative observations showed good

cell adhesion and proliferation of KCs on the PNIPAAm coated membranes in both sections of the

study. The rapid cell detachment, which was seen in Chapter 2.2c, being attributed to the membranes

porous nature. Transfer of the cell sheets was the main challenge of our study. The plastic barrier

around the insert made the removal of the membrane a demanding process. For future studies it would

be advantageous to remove the inserts circular border after spin coating with PNIPAAm. The use of a

slightly flexible “transport surface” to transfer the KCs from the coated membrane to a desired final

location, would also be an improvement on our current experimental design. Maneuvering the

membrane when it is moist is currently very challenging, so the use of this intermediary would improve

the efficiency of the process. This solution would be analogous to Pasuwat et al's hydrophilically

modified (PVDF) transfer membrane, which allowed the in vitro transfer of chondrocyte cell sheets

[21].

The second section of the study also produced promising results, as the trend that was observed

via caspase-3 staining did not show a difference between KCs cultured on PNIPAAm coated and

uncoated surface. Here we would like to extend the study to quantitatively look at KC proliferation on

33

all three surfaces (uncoated dishes, coated dishes, coated membranes). Qualitative analysis from the

experiments conducted thus far show superior cell growth on our PNIPAAm coated membranes.

After the completion of the above modifications, experiments incorporating an animal model will

be more manageable. Our group has considerable experience studying the diabetic wound healing

process in the rat animal model [61], [62]. Fusing our current KC cell sheet work with the previous in

vivo wound healing studies, would prove a huge step forward towards the creation of a therapeutic

smart band aid for the treatment of diabetic wounds, as illustrated earlier in Figure 2.1. Hopefully, the

PNIPAAm coated membranes cell compatibility is not limited to KC, as we can name a multiple other

therapeutic applications from Chapter 1 alone where improved cell viability and increased cell release

would be greatly advantageous.

34

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