Shinshu Med J, 72⑶：149～157, 2024

Cytocompatibility of Lactic Acid-extracted Barramundi
Skin Collagen for Cell Culture Applications
Yali Men1）, Chuang Ma2）3）, Daihachiro Tomotsune1）2）
Fengming Yue1）2）＊ and Kohei Johkura1）＊
1） Department of Histology and Embryology, Shinshu University School of Medicine
2） Shinshu University Interdisciplinary Cluster for Cutting Edge Research, Institute for Biomedical Sciences
3） Biomedical Engineering Division, Graduate School of Medicine, Science and Technology, Shinshu University

Fish collagen is becoming a potent material in many fields including the biomedical field by substituting fordomestic animals that may cause zoonotic diseases. In this study, the skin of farmed barramundi (Lates calcarifer)
was employed as a collagen material, and the affinity of this collagen for live cells was assessed. Four extraction
solutions for the skin collagen were compared : acetic acid with/without pepsin as conventionally used, and alter­
natively lactic acid with/without pepsin. Among the resulting collagens, lactic acid-extracted collagen (LC) showeda meshwork of relatively fine fibers after lyophilization in scanning electron microscopy and a hydroxyproline
content of 7.7 ％ in amino acid analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
showed that this skin collagen was type I with α1 and α2 chains, and these chains had relatively close molecularweight as compared with those of porcine collagen. The viability of mouse fibroblasts exposed to LC at various
concentrations for 24 hours was maintained at ≥ 93 ％, demonstrating its low cytotoxicity. In a wound healing assay
using each collagen in the culture medium, LC supported more active motility of mouse fibroblasts than colla­
gens obtained by the other extraction methods. Collectively, these results suggest that barramundi skin collagenextracted by lactic acid has suitable properties in cellular activities, exhibiting advantages over the conventional
methods, and is expected to be a useful material for cell culture applications. Shinshu Med J 72 : 149―157, 2024
(Received for publication January 16, 2024 ; accepted in revised form February 26, 2024)
Key words : fish skin, collagen, lactic acid extraction, cellular response

Ｉ Introduction

and plays an important role as a signaling moleculethat directly or indirectly controls cell activities, in­Many materials that originate from collagen, suchcluding differentiation, wound healing, and blood clot­

as collagen gels, gelatin, and collagen peptides, areting1）-3）. Such biological activities are mediated by thewidely used as cell culture substrates in many researchinteraction between collagen and biopolymers suchfields. Collagen functions as a framework for tissues

as proteins and polysaccharides. Dozens of collagenbinding proteins have been identified, and the role of＊Corresponding author : Fengming Yue
Department of Histology and Embryology,
Shinshu University School of Medicine,
3-1-1 Asahi, Matsumoto, Nagano 390-8621 Japan＊

collagen as a signaling molecule is becoming evident4）.
The use of natural collagen is increasing in researchin the biomedical field2）. Collagen is also marketed as

E-mail : yuerat@shinshu-u.ac.jp

a food additive, health functional food, and functional

Corresponding author : Kohei Johkura

cosmetic. Most raw materials for collagen production

Department of Histology and Embryology,
Shinshu University School of Medicine,
3-1-1 Asahi, Matsumoto, Nagano, 390-8621 Japan
E-mail : kohei@shinshu-u.ac.jp

No. 3, 2024

are obtained from the bones or skin of domestic ani­mals, such as bovine and porcine, which have highresources and yields. However, there is a risk of zoo­
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Men・Ma・Tomotsune et al.

notic diseases, such as bovine spongiform encepha­[1 : 20 (m/v)] for 36 hours to remove impurities andlopathy and foot-and-mouth disease, which became apigments. The alkali-treated fish skin was rinsed

5）6）. Additionally, somewith deionized water to achieve neutrality and thenpeople have cultural issues that restrict the handlingsoaked in four acidic solutions [0.5 M lactic acid, 0.5

of mammalian products. For these reasons, the search

M acetic acid, 0.5 M lactic acid containing 1 g/100

for raw materials to replace mammalian tissues has

mL pepsin (Fujifilm Wako, Osaka, Japan), and 0.5 M

become a challenge. Therefore, attention has been

acetic acid containing 1 g/100 mL pepsin] while stirred

focused on fish with similar materials and few zoo­in a stirrer (300 rpm) for 24 hours to extract collagen.

notic diseases. This replacement has been progress­

The resulting solution was centrifuged at 10,000×g

hot topic in 1990s to early 2000s

7）

for 20 minutes at 4 ℃. The supernatant was collected

In this context, we focused on fish skin. The fish

and thoroughly precipitated with salt to a final con­skin is rich in collagen and other major components

centration of 0.9 M so that remnant pepsin was re­that comprise the body. In particular, when consider­

moved. The sample after salting out was centrifuged

ing the diversity of their functions, proteins unique

at 10,000×g, for 15 minutes at 4 ℃, and the precipi­

to fish skin possibly exist, and they have emerged astate was collected. The collected precipitate wasattractive materials that warrant marketization andcompletely dissolved in a 0.5 M lactic acid or acetic

research. In this study, we used skin from barramundiacid solution, followed by dialysis for 24 hours in a

(Lates calcarifer). This is a large fish reaching a length

0.1 M lactic acid or acetic acid solution [1 : 50 (v/v)],of 2 m, inhabiting the tropics of Indian and Pacificand then dialyzed in deionized water of the same

Oceans, and is farmed in various places in Southeast

volume for 72 hours. The dialyzed sample was freeze

Asia for food. Barramundi skin is discarded and pro­dried to obtain the extracted collagen. All operations

duces enormous amounts of waste, and is therefore

were carried out at 4 ℃. The four kinds of collagen

inexpensive. Moreover, this skin has been scarcely

extracted by the various acidic solutions, lactic acid-reported8）, and has not been practically used. Thus, it

extracted collagen (LC), acetic acid-extracted colla­

is expected that new materials will be discovered andgen (AC), lactic acid-pepsin-extracted collagen (LPC),

applied.

and acetic acid-pepsin-extracted collagen (APC), were

ing rapidly in recent years .

To search for new materials applicable for devel­
opment in the biomedical field, we evaluated the suit­subjected to analyses.
Ｃ

SEM Observation

ability of barramundi skin collagen for live cells.

The morphology of freeze-dried collagen was ob­

Among four extraction methods assessed, a method

served by scanning electron microscopy (SEM ; JSM-

using lactic acid was found to yield collagen in a rea­

7600F, JEOL, Tokyo, Japan). Collagen samples were

sonably natural form. This collagen showed uniqueattached to an adhesive carbon stub and coated withfeatures and favorable effects on cell activities in vitro.

gold. A SEM was used for imaging at a voltage of 15

Ⅱ
Ａ

Materials and MethodskV.
Ｄ

SDS-PAGE analysis
The chain compositions of collagens were analyzed

Materials
Barramundi skin was purchased from Qihai (Zhuji­

by 8 ％ SDS-PAGE under reducing condition. Eachang, China).

collagen sample was dissolved in diluted HCl (pH 3)

Ｂ

at a concentration of 3 mg/mL. A 21 µl collagen solu­

Collagen extraction from fish skin
Collagen was extracted from barramundi skin using
9）-11）

tion was mixed with 7 µl of 4×Laemmli sample buf­with some modi­

fer containing 2-mercaptoethanol (Bio-Rad, CA,

fications. Briefly, the fish skin was cut into smallUSA). The mixed solution was boiled for 5 min anda previously established method2pieces measuring 0.5 cm using scissors. Subsequently,

then loaded onto the gel. Electrophoresis was per­the pieces were immersed in a 0.1 M NaOH solution

formed at 80 V for 90 min. After electrophoresis, the150

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Barramundi skin collagen : a potential material for cell culture

gel was stained with Coomassie brilliant blue, fol­Ｈ

Statistical analysis

lowed by de-staining in a solution of 10 ％ (v/v) metha­

Statistical analysis of cytotoxicity was performed

nol and 10 ％ (v/v) acetic acid. The stained gel wasusing Dunnett’s multiple comparisons test. P＜0.05

imaged by a scanner.

was considered significant.

Ｅ

Amino acid composition analysis of collagen

Ⅲ Results

After hydrolysis in hydrochloric acid, the collagensample was sealed in a digestion tube and subjectedＡ Light and electron microscopic observations of

to vacuum treatment at 110 ℃. Subsequently, high-collagensperformance liquid chromatography was employed

When barramundi skin collagens extracted by thefor analysis at The Food Technology Department offour methods were lyophilized, they had a white

the Nagano Prefectural Industrial Technology Centercotton-like appearance under macroscopic observa­(Nagano, Japan). The results are expressed as thetion (Fig. 1A-D). These collagens exhibited a com­

number of specific amino acid residues per 1,000 amino

plex meshwork consisting of filamentous and film-

acid residues.

like fibers in light microscopy (Fig. 1E-H). In SEM,Ｆ

the meshwork fibers had different shapes, pore sizes,

Cytotoxicity assay
A cytotoxicity assay of the extracted collagens wasand arrangements (Fig. 2). LC had more fine andperformed by evaluating the cell viability of mouse

regular filaments than AC (Fig. 2A, B). Addition offibroblasts (SNL ; ATCC, USA) after exposure to each

pepsin to each acid extraction formed fibers enrichedcollagen at 0.5-2.0 mg/mL for 24 hours. SNL cellswith film-like structures (Fig. 2C, D).

were seeded in 96-well plates at a density of 1×10

5Ｂ

Amino acid composition analysis

cells/mL and cultured in high glucose Dulbecco’s

Table 1 shows the results of amino acid analysis ofModified Eagle Medium (Fujifilm Wako) containing

LC and AC. In terms of the contents of the three

10 ％ fetal bovine serum (FBS ; Gibco, MO, USA).

amino acids that characterize collagen molecules, LC

After 24 hours of culture, a collagen solution (0.5-2.0

was 34.4 ％ glycine, 11.7 ％ proline, and 7.7 ％ hy­

mg/mL) was applied to the culture for an additional

droxyproline. These values were close to those of AC.

®

24 hours. Cell viability was assayed using alamarBlue

Because the glycine content was almost one third ofreagent (Bio-Rad).

the total amino acids, and hydroxyproline that nearlyＧexclusively exists in collagen was detected to a cer­Wound healing assay
5

SNL cells were cultured in a 12-well plate (1.5×10

tain extent, the extraction was successful and nearlycells/well) to confluency (24 h). This single layer ofpure collagen was extracted.

cells was scratched with a 200 µL pipette tip. TheＣ

SDS-PAGE analysis of collagens

medium was aspirated, and the cells were washed

The type of barramundi skin collagen was exam­with Dulbecco’s phosphate-buffered saline (DPBS ;ined by SDS-PAGE (Fig. 3). Two bands around 120

Fujifilm Wako). Then, medium with 2 ％ FBS and 1.5

kDa indicating α chains were most discernible in LC.

mg/mL collagen extract was applied to the cells.

This suggested that the barramundi skin collagenThis concentration of collagen was determined ac­

was mainly type I with a heterotrimeric structure ofcording to preliminary study that examined various

α1 and α2 chains, which constituted the control ofconcentrations based on the results of cytotoxicity

porcine skin collagen. The molecular weight differ­assay. The appearance of cells migrating and fillingence between α1 and α2 was less than 5 kDa in LC,

the defect was observed under a phase-contrast mi­

which was smaller than that in porcine type I colla­

croscope (BZ-X710 ; KEYENCE, Osaka, Japan) imme­

gen with a difference of ≥ 10 kDa. The band around

diately after the collagen had been applied and then

120 kDa appeared to be nearly single for AC, LPC,

24 hours later.

and APC, which may be due to the proximity andamount of the two α chains.

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Fig. 1 Macroscopic (A-D) and light microscopic (E-H) observations of lyophilized collagens extracted frombarramundi skin by four methods : LC (A, E), AC (B, F), LPC (C, G), and APC (D, H). Each collagen exhib­
ited a meshwork of fibers with various appearances. E-H : ×80.

Fig. 2 SEM observation of the meshwork of LC (A), AC (B), LPC (C), and APC (D). Note the relatively
fine and regular fibers in LC (A) compared with AC (B). Film-like strands were abundant in
LPC and APC (C, D). Bars＝100 µm.

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Barramundi skin collagen : a potential material for cell culture

Fig. 3 SDS-PAGE analysis of extracted
collagens
Lane 1 : molecular weight marker. Lane 2 :control (porcine skin collagen). Lane 3 : LC,
Lane 4 : AC, Lane 5 : LPC, and Lane 6 : APC.
LC showed a split band or close bands of α1and α2 chains. The molecular weight differ­ence between the two α chains was relatively
small. The difference in porcine skin collagen
was approximately ≥ 10 kDa. The β chain
around 240 kDa was two α chains connectedby crosslinking.

Ｄ Cytotoxicity assay of the collagens in mouse

Table 1

fibroblasts
In the cytotoxicity assay, mouse fibroblasts were
exposed to each collagen at concentrations of 0.5-2.0

Amino acid

Amino acid analysis

Number of residues(/1000 residues)

mg/mL for 24 hours, and then cell viability was eval­

LC

AC

Asp

46

46

uated (Fig. 4). LC exposure resulted in no significant

Thr

23

23

reduction in viability at all concentrations, which was

Ser

26

26

Glu

73

72

Gly

344

347

Ala

137

137

Val

21

21

Ile

10

9

Leu

20

19

Tyr

3

2

Phe

17

15

Lys

27

27

The motility of mouse fibroblasts was assessed by a

His

6

6

wound healing assay in medium containing each colla­

Arg

53

53

Hyp

77

83

Pro

117

115

total

1000

1000

maintained at ≥ 93 ％ (Fig. 4A). A slight but significant
reduction in viability was evident after AC exposure(Fig. 4B). LPC and APC also significantly reduced
cell viability to approximately 80 ％ (Fig. 4C, D).
These results showed that LC did not remarkably
affect cell viability and had low cytotoxicity in mouse
fibroblasts.
Ｅ

Wound healing assay of mouse fibroblasts

gen at 1.5 mg/mL (Fig. 5). LC induced more active
cell migration than AC, and the cell defect area wascompletely filled after 24 hours (Fig. 5A-D). Also, thedefect area was favorably filled after exposure to LPC,
but not APC (Fig. 5E-H). LC and even LPC were
demonstrated to promote active motility of mouse fi­

Amino acid analysis of LC and AC revealed a highcontent of hydroxyproline (Hyp), which almost exclu­
sively exists in collagens among proteins.

broblasts.
Ⅳ Discussion

terized by a repeated amino acid triplet, glycine-X-Y,
in which X and Y are frequently proline and hydroxy­

Collagen has, at least partially, a helical structure

proline, respectively. The α chain is subclassified into

composed of three α chains. The single α chain with a

α1 and α2 chains by slight differences in the amino

molecular weight of approximately 100,000 is charac­acid composition, and they maintain the triple helix

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Fig. 4 Viability of cells exposed to the extracted collagens
Mouse fibroblasts were exposed to each extracted collagen at concentrations of 0.5-2.0 mg/mL for 24 hours,
and then cell viability was evaluated by alamarBlue® reagent. (A) LC maintained cells viability at ≥ 93 ％without a significant decline compared with the control. (B) Cell viability under AC exposure was similar tothat under LC exposure, but the decline was statistically significant at concentrations of 1.0 and 1.5 mg/mL.
(C, D) Cell viability was significantly reduced by LPC and APC to approximately 80 ％. Control : no collagen
exposure. Data are expressed as the mean±SD (n＝4) ; ＊p＜0.05, ＊＊p＜0.01.

Fig. 5 Effect of the barramundi skin collagens on wound healing of mouse fibroblasts
Confluent fibroblasts with a linear scratch defect were cultured for 24 hours in medium containing 1.5 mg/mL
of LC (A, B), AC (C, D), LPC (E, F), or APC (G, H). The defect area was fully covered by migrated cells after 24hours of LC exposure (A, B), but remained after AC exposure (C, D). Similar results were observed for LPC (E,F) and APC (G, H). Dotted lines indicate the edge of cell layers or the front line of migrating cells. Bars＝1 mm.
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Barramundi skin collagen : a potential material for cell culture

structure unique to collagen12）. Twenty-nine types oftime-consuming and costly11）. The extraction method

collagen molecules have been reported, depending on

using lactic acid alone in this study has advantages

13）

the structure . Most collagen is type I with a het­over the conventional techniques because of simplicity

erotrimer of two α1 chains and a single α2 chain (α1×and low cost.

2, α2×1), but homotrimer collagens composed of

The lyophilized collagens from barramundi skinthree α1 chains (α1×3) exist, such as type II and III

showed variable aspects under four extraction condi­

14）

collagens . Homotrimeric type III collagen promotes

tions (Fig. 1, 2). LC had a relatively fine meshwork

self-assembly to fibrils compared with heterotrimericcompared with AC, while LPC and APC showed film-

15）

type I collagen . Among the collagens, types I-IV
13）

like fibers. A previous study reported that collagenentanglement depends strongly on the type of acidare mainly used as biomaterials .

Barramundi skin collagen as LC had both α1 andused, and the telopeptides regulate the properties of

α2 chains, which suggested type I collagen with thecollagen molecules18）. Native collagen with the telo­

α1×2 and α2×1 structure (Fig. 3). The difference inpeptides showed finely meshed amorphous structures,the molecular weights of α1 and α2 chains was smaller

whereas telopeptide-poor collagen formed crystal­than that of porcine type I collagen. The amino acidline-like and well-ordered structures18）. In addition,

composition, particularly the hydroxyproline content,

the viscosity of collagen solution seems to influence

determines the denaturation temperature of collagen,the meshwork structure18）. The viscosity of collagenat which the triple helix dissociates to the monomer

solution in lactic acid was reported to be lower than13）

and changes to gelatin . Thus, the higher the content,

that in acetic acid at 25 ℃19）. In our study, there may

the more thermally stable collagen gel is formed when

have been some difference in the viscosity of the col­applied to a cell culture environment. In the present

lagen solution even after dialysis. Therefore, it isstudy, the hydroxyproline content of barramundi

supposed that the deletion of telopeptide by pepsin,skin collagen was 7.7 ％ in LC (Table 1). In previous

as well as the differential effects of lactic acid andreports on collagens from various barramundi tissues,acetic acid on collagen solutions, alters the physico­the denaturation temperature and hydroxyproline con­chemical properties of collagen and explains the16）tent (in parentheses) were 36.4 ℃ (8.4 ％) in scales ,35.0 ℃ (8.3 ％) in the swim bladder, and 33.3 ℃ (7.9 ％)8）structural variation associated with lyophilization.
Because LC exhibited better results in cell viability

in skin , which are at the boundary with terrestrial

and motility assays than AC (Fig. 4, 5), LC seems toanimals that have been used so far and close to hu­

be useful when applied to cell culture. When applica­17）

man body temperature . LC from barramundi skintion to living body is considered, however, LPC may

in our study was supposed to have a similar denatur­

have some benefit due to the absence of telopeptides.

ation temperature to these reports as judged from

Although the cell viability was inferior to LC, LPC

the hydroxyproline content, though the content will

showed favorable result next to LC in the wound

differ depending on extraction condition. It may havehealing assay. Anyway, the issue of antigenicity ofgood affinity for human cells.

acid-extracted collagen still remains and should be

In conventional collagen purification methods, anevaluated and resolved.

enzyme such as pepsin is added to hydrolyze collagen

The limitations of this study can be considered as

for a long time, combining acid solubilization if neces­follows. Collagen coating was not employed for the

10）
11）

. The enzyme treatment reduces the antige­

wound healing assay. The collagens were directly

nicity of collagen by deleting telopeptides at both

added into the culture medium to confirm their effectends of the triple helix and is efficient to obtain colla­

on cell motility at the same concentration of 1.5 mg/

gen applicable to humans. However, since treatment

mL used in the cytotoxicity assay. Moreover, sincewith a protease inhibitor may be employed to inacti­

these experiments were conducted using mouse fi­vate the enzyme, conventional methods are generally

broblasts, they cannot be applied directly to humansary

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Men・Ma・Tomotsune et al.

cells. Considering the potential application to humanramundi skin collagen can be used for various purpos-

cells and bodies, the effect of barramundi skin colla-

es, such as cell culture additives, 3D culture substrates,

gens on human fibroblasts should be further con-and scaffolding materials. This study contributes tofirmed by assessing cell viability and cell motility on

the practical application of fish collagen in the bio-

collagen-coated surface in further studies.

medical field.

Our method for collagen extraction from barramundi

Acknowledgment

skin converts unused waste into valuable raw materials
with a reasonable price and quality. This has an ad-

We would like to thank the Division of Instrumental

vantage in the development and manufacture of col-Research, Shinshu University Research Center forlagen raw materials and products applicable to cells,

Advanced Science and Technology for the support

being suitable for a recycling-oriented society. Bar-on SEM analysis.

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（2024. 1. 16 received ; 2024. 2. 26 accepted）

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