Cancer Cell Culture Methods and Protocols

Methods in Molecular Biology 2645 Dania Movia

(Editor) download

https://ebookmeta.com/product/cancer-cell-culture-methods-and-
protocols-methods-in-molecular-biology-2645-dania-movia-editor/

Download more ebook from https://ebookmeta.com

We believe these products will be a great fit for you. Click
the link to download now, or visit ebookmeta.com
to discover even more!

Cancer Cell Biology Methods and Protocols Methods in

Molecular Biology 2508 Sherri L. Christian (Editor)

https://ebookmeta.com/product/cancer-cell-biology-methods-and-
protocols-methods-in-molecular-biology-2508-sherri-l-christian-
editor/

Mouse Cell Culture Methods and Protocols Methods in

Molecular Biology 633 Andrew Ward

https://ebookmeta.com/product/mouse-cell-culture-methods-and-
protocols-methods-in-molecular-biology-633-andrew-ward/

Cell Viability Assays Methods and Protocols Methods in

Molecular Biology 2644 Oliver Friedrich (Editor)

https://ebookmeta.com/product/cell-viability-assays-methods-and-
protocols-methods-in-molecular-biology-2644-oliver-friedrich-
editor/

Litigating Transnational Human Rights Obligations

Alternative Judgments 1st Edition Mark Gibney Editor
Wouter Vandenhole Editor

https://ebookmeta.com/product/litigating-transnational-human-
rights-obligations-alternative-judgments-1st-edition-mark-gibney-
editor-wouter-vandenhole-editor/
Cold Cases Solved Volume 5 18 Fascinating True Crime
Cases 1st Edition Robert Keller

https://ebookmeta.com/product/cold-cases-solved-
volume-5-18-fascinating-true-crime-cases-1st-edition-robert-
keller/

Evolutionary Psychiatry Current Perspectives on

Evolution and Mental Health 1st Edition Riadh Abed

https://ebookmeta.com/product/evolutionary-psychiatry-current-
perspectives-on-evolution-and-mental-health-1st-edition-riadh-
abed/

Operations Management 10th Edition Nigel Slack

https://ebookmeta.com/product/operations-management-10th-edition-
nigel-slack/

Epistemic Dilemmas New Arguments New Angles 1st Edition

Kevin Mccain Editor Scott Stapleford Editor Matthias
Steup Editor

https://ebookmeta.com/product/epistemic-dilemmas-new-arguments-
new-angles-1st-edition-kevin-mccain-editor-scott-stapleford-
editor-matthias-steup-editor/

The Care of the Older Person 5th Edition Ronald Caplan

https://ebookmeta.com/product/the-care-of-the-older-person-5th-
edition-ronald-caplan/
Physical Chemistry Thermodynamics Structure and Change
Peter Atkins Julio de Paula Tenth 2014 10th Edition
Peter Atkins

https://ebookmeta.com/product/physical-chemistry-thermodynamics-
structure-and-change-peter-atkins-julio-de-paula-tenth-2014-10th-
edition-peter-atkins/
Methods in
Molecular Biology 2645

Dania Movia
Adriele Prina-Mello
Editors

Cancer Cell
Culture
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Cancer Cell Culture

Methods and Protocols

Edited by

Dania Movia and Adriele Prina-Mello

Trinity Translational Medicine Institute, Trinity Centre for Health Sciences, Dublin, Ireland
Editors
Dania Movia Adriele Prina-Mello
Trinity Translational Medicine Institute Trinity Translational Medicine Institute
Trinity Centre for Health Sciences Trinity Centre for Health Sciences
Dublin, Ireland Dublin, Ireland

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-3055-6 ISBN 978-1-0716-3056-3 (eBook)
https://doi.org/10.1007/978-1-0716-3056-3
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover image courtesy of Despina Bazou.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

In the past ten years, cancer research has been advancing at a fast pace with the discoveries of
the influence of the tumor microenvironment on tumor growth and progression, of new
associated biomarkers, and the subsequent development of new in vitro experimental
research models. These have enabled the investigation of biochemical and molecular path-
ways in a mechanistic way with accuracy and precision previously unknown, thus enabling
the discovery of new targeted drugs and immunotherapies. A continuous need for standar-
dized in vitro methods for cancer cell culture has however emerged, thus thriving to achieve
precision targeting, accelerated drug development, and predictive efficacy assessment of new
treatments.
The intention behind this book is to provide researchers with a collection of cell models
that can be used for preclinical cancer research. After an initial introductory overview, the
methodological chapters describe both conventional two-dimensional (2D) and more inno-
vative three-dimensional (3D) culturing techniques, and how these can be deployed to
address specific cancer types, or subtypes, or to recapitulate the interaction complexity
between cancer cells and treatments.
This book is therefore divided into two parts. The first part includes introductory
chapters, with historical and chronological mini-review type chapters which present the
state-of-the-art in preclinical cancer research, from the basics of 2D cancer cell culture, to
the cell models at the air-liquid interface, and finally moving on to the most recent
advancements in the development of 3D complex spheroid models and dedicated disease
animal models.
Following that, the second part of the book is dedicated to the technical chapters, which
illustrate step-by-step methodologies for specific cancer cell culture methods. These chap-
ters have been written by contributors with extensive expertise, and who use these models in
their laboratories for preclinical cancer research. Methods range from the generation of
isogenic cancer cell lines, and the use of serum-free growth conditions, to the formation of
3D cell cultures and the use of specific assays for the efficacy assessment of new anticancer
therapies.
In conclusion, the overall value of this book on in vitro cancer cell methods lies on the
practical experimental step-by-step knowledge transfer provided by the contributing
authors, their extensive knowledge sharing in the presented models, and the ability to
connect with them for assistance going forward in the adoption and use of any of the
presented models.
Therefore, with such invaluable contributions, we hope that every specific contribution
here presented can represent the starting point for further improved and advanced models
to be developed by the community of researchers involved in creating a breakthrough in
cancer research, whether through the development of new treatments or through the
creation of new models for getting insight on unknown cancer pathophysiology
mechanisms.

Dublin, Ireland Dania Movia

Adriele Prina-Mello

v
Acknowledgments

We are grateful to the authors of the various chapters, for their expert input and constructive
cooperation in the timely preparation of this book. We also extend our thanks to Prof. John
M. Walker, as Series Editor.
We gratefully acknowledge the partial financial support of Science Foundation Ireland
and the Irish Research Council (SFI-IRC Pathway Programme to D.M.), and European
Union under the Horizon 2020 (NoCanTher (grant 685795), Safe-N-Medtech (grant
814607), and Expert (grant 825828) projects) and Horizon-Europe (INSPIRE project)
programs.
It is with genuine gratitude and warm regard that we dedicate this book in memory of
Prof. Yuri Volkov, immunologist, cancer researcher, and mentor, who has meant and
continues to mean so much to us. Although he is no longer among us, his memory
continues to inspire our research.

Dania Movia
Adriele Prina-Mello

vii
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I OVERVIEW OF THE EXISTING METHODS FOR CANCER

CELL CULTURE
1 Cancer Cell Culture: The Basics and Two-Dimensional Cultures . . . . . . . . . . . . . 3
Melissa Anne Tutty, Sarah Holmes, and Adriele Prina-Mello
2 Cell Cultures at the Air–Liquid Interface and Their Application
in Cancer Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Luisana Di Cristo and Stefania Sabella
3 Three-Dimensional Spheroids for Cancer Research. . . . . . . . . . . . . . . . . . . . . . . . . . 65
Melissa Anne Tutty and Adriele Prina-Mello
4 Disease Animal Models for Cancer Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Sara Fuochi and Viola Galligioni

PART II SPECIFIC METHODS FOR CANCER CELL CULTURE:

STEP-BY-STEP METHODOLOGIES
5 Generation of Radioresistant Prostate Cancer Cells. . . . . . . . . . . . . . . . . . . . . . . . . . 129
Laure Marignol
6 Generation and Characterization of an Isogenic Cell Line Model
of Radioresistant Esophageal Adenocarcinoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Aoife Cannon, Stephen G. Maher, and Niamh Lynam-Lennon
7 Developing an In Vitro Isogenic Model of Chemotherapy-Resistant
Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Martin P. Barr
8 Culturing Human Lung Adenocarcinoma Cells in a Serum-Free
Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Aline Chary
9 A Method for Culturing 3D Tumoroids of Lung Adenocarcinoma
Cells at the Air–Liquid Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Dania Movia and Adriele Prina-Mello
10 Isolation and Cryopreservation of Mononuclear Cells from
Peripheral Blood and Bone Marrow of Blood Cancer Patients . . . . . . . . . . . . . . . . 179
Sarah Brophy, Rebecca Amet, Hayley Foy-Stones,
Nicola Gardiner, and Anthony M. McElligott
11 Serum-Free Production of Human Stem Cell-Derived Liver
Spheres for Cancer Metastasis Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Alvile Kasarinaite, James Drew, Mantas Jonaitis, Elaine Ma,
Laura M. Machesky, and David C. Hay

ix
x Contents

12 A Self-Assembly Method for Creating Vascularized Tumor

Explants Using Biomaterials for 3D Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Lance L. Munn and Despina Bazou
13 A Step-by-Step Methodological Guide for Developing Zonal
Multicellular Scaffold-Based Pancreatic Cancer Models . . . . . . . . . . . . . . . . . . . . . . 221
Priyanka Gupta and Eirini G. Velliou
14 Measuring Immune Cell Movement Toward the Soluble Microenvironment
of Human Tissues Using a Boyden Chamber-Based Migration Assay . . . . . . . . . . 231
Eimear Mylod, Joanne Lysaght, and Melissa J. Conroy
15 A Method for the In Vitro Cytotoxicity Assessment of Anti-cancer
Compounds and Materials Using High Content Screening Analysis. . . . . . . . . . . 241
Melissa Anne Tutty and Adriele Prina-Mello
16 Testing the Effects of Magnetic Hyperthermia in 2D Cell Culture . . . . . . . . . . . . 251
Gary Hannon and Adriele Prina-Mello
17 Cell Viability Assay with 3D Prostate Tumor Spheroids. . . . . . . . . . . . . . . . . . . . . . 263
Ezgi Oner, Steven G. Gray, and Stephen P. Finn
18 Analysis of Cancer Cell Line Secretomes: A Complementary Source
of Disease-Specific Protein Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Katie Dunphy, Despina Bazou, and Paul Dowling

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Contributors

REBECCA AMET • The John Durkan Leukaemia Laboratory, Trinity Translational Medicine
Institute, Trinity College and St James’s Hospital, Dublin, Ireland
MARTIN P. BARR • Thoracic Oncology Research Group, Trinity St James’s Cancer Institute,
Trinity Translational Medicine Institute, St James’s Hospital & Trinity College Dublin,
Dublin, Ireland
DESPINA BAZOU • Department of Haematology, Mater Misericordiae University Hospital,
Dublin, Ireland; School of Medicine, University College Dublin, Dublin 4, Ireland
SARAH BROPHY • The John Durkan Leukaemia Laboratory, Trinity Translational Medicine
Institute, Trinity College and St James’s Hospital, Dublin, Ireland
AOIFE CANNON • Department of Surgery, Trinity St. James’s Cancer Institute, Trinity
Translational Medicine Institute, Dublin, Ireland
ALINE CHARY • Environmental Research and Innovation (ERIN) Department,
Luxembourg Institute of Science and Technology (LIST), Esch-sur-Alzette, Luxembourg
MELISSA J. CONROY • Cancer Immunology Research Group, Department of Physiology, School
of Medicine, Trinity College Dublin, Dublin, Ireland
LUISANA DI CRISTO • D3 PharmaChemistry, Nanoregulatory Group, Italian Institute of
Technology, Genoa, Italy
PAUL DOWLING • Department of Biology, Maynooth University, National University of
Ireland, Kildare, Ireland
JAMES DREW • CRUK Beatson Institute, Glasgow, UK
KATIE DUNPHY • Department of Biology, Maynooth University, National University of
Ireland, Kildare, Ireland
STEPHEN P. FINN • Thoracic Oncology Research Group, Trinity Translational Medicine
Institute, St. James’s Hospital, Dublin, Ireland; Department of Clinical Medicine, Trinity
College Dublin, Dublin, Ireland; Department of Histopathology and Morbid Anatomy, Sir
Patrick Dun Translational Research Lab, St. James’s Hospital, Dublin, Ireland;
Department of Histopathology, Labmed Directorate, St. James’s Hospital, Dublin, Ireland;
Cancer Molecular Diagnostics, Labmed Directorate, St. James’s Hospital, Dublin, Ireland
HAYLEY FOY-STONES • Cryobiology Laboratory Stem Cell Facility, St James’s Hospital,
Dublin, Ireland
SARA FUOCHI • Universit€ a t Bern, Experimental Animal Center, Bern, Switzerland
VIOLA GALLIGIONI • Netherlands Institute for Neuroscience - KNAW, Amsterdam, The
Netherlands
NICOLA GARDINER • Cryobiology Laboratory Stem Cell Facility, St James’s Hospital, Dublin,
Ireland
STEVEN G. GRAY • Thoracic Oncology Research Group, Trinity Translational Medicine
Institute, St. James’s Hospital, Dublin, Ireland; Department of Clinical Medicine, Trinity
College Dublin, Dublin, Ireland
PRIYANKA GUPTA • Centre for 3D models of Health and Disease, Division of Surgery and
Interventional Science, University College London, London, UK
GARY HANNON • Laboratory of Biological Characterization of Advanced Materials
(LBCAM), Trinity Translational Medicine Institute, Trinity College Dublin, Dublin,

xi
xii Contributors

Ireland; Trinity St. James’s Cancer Institute, St. James’s Hospital, Trinity College Dublin,
Dublin, Ireland
DAVID C. HAY • Centre for Regenerative Medicine, University of Edinburgh, Edinburgh,
UK
SARAH HOLMES • Laboratory of Biological Characterization of Advanced Materials
(LBCAM), Trinity Translational Medicine Institute, Trinity College, Dublin, Ireland
MANTAS JONAITIS • Centre for Regenerative Medicine, University of Edinburgh, Edinburgh,
UK
ALVILE KASARINAITE • Centre for Regenerative Medicine, University of Edinburgh,
Edinburgh, UK
NIAMH LYNAM-LENNON • Department of Surgery, Trinity St. James’s Cancer Institute,
Trinity Translational Medicine Institute, Dublin, Ireland
JOANNE LYSAGHT • Cancer Immunology and Immunotherapy Group, Department of
Surgery, Trinity Translational Medicine Institute, St James’s Hospital, Dublin, Ireland
ELAINE MA • CRUK Beatson Institute, Glasgow, UK; Institute of Cancer Sciences,
University of Glasgow, Glasgow, UK
LAURA M. MACHESKY • CRUK Beatson Institute, Glasgow, UK; Institute of Cancer Sciences,
University of Glasgow, Glasgow, UK
STEPHEN G. MAHER • Department of Surgery, Trinity St. James’s Cancer Institute, Trinity
Translational Medicine Institute, Dublin, Ireland
LAURE MARIGNOL • Translational Radiobiology and Oncology Group, Applied Radiation
Therapy Trinity Research Group, Trinity College Dublin, Dublin, Ireland
ANTHONY M. MCELLIGOTT • The John Durkan Leukaemia Laboratory, Trinity
Translational Medicine Institute, Trinity College and St James’s Hospital, Dublin,
Ireland; Trinity St. James’s Cancer Institute, Trinity College and St James’s Hospital,
Dublin, Ireland
DANIA MOVIA • Applied Radiation Therapy Trinity (ARTT), Discipline of Radiation
Therapy, Trinity Centre for Health Sciences, Trinity College Dublin, Dublin, Ireland;
Laboratory for Biological Characterisation of Advanced Materials (LBCAM), Trinity
Translational Medicine Institute, Trinity Centre for Health Sciences, Trinity College
Dublin, Dublin, Ireland; Trinity St. James’s Cancer Institute, St. James’s Hospital, Trinity
College Dublin, Dublin, Ireland
LANCE L. MUNN • Edwin L. Steele Laboratory, Department of Radiation Oncology,
Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
EIMEAR MYLOD • Cancer Immunology and Immunotherapy Group, Department of Surgery,
Trinity Translational Medicine Institute, St James’s Hospital, Dublin, Ireland
EZGI ONER • Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Izmir
Katip Celebi University, Balatcik, Izmir, Turkey; Thoracic Oncology Research Group,
Trinity Translational Medicine Institute, St. James’s Hospital, Dublin, Ireland;
Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland
ADRIELE PRINA-MELLO • Laboratory for Biological Characterization of Advanced Materials
(LBCAM), Trinity Translational Medicine Institute, Trinity Centre for Health Sciences,
Trinity College Dublin, Dublin, Ireland; Nanomedicine and Molecular Imaging Group,
Trinity Translational Medicine Institute, (TTMI), School of Medicine, Trinity College
Dublin, Dublin, Ireland; Trinity St. James’s Cancer Institute, St. James’s Hospital, Trinity
 Contributors xiii

College Dublin, Dublin, Ireland; Advanced Materials and Bioengineering Research

(AMBER) Centre, CRANN Institute, Trinity College Dublin, Dublin, Ireland
STEFANIA SABELLA • D3 PharmaChemistry, Nanoregulatory Group, Italian Institute of
Technology, Genoa, Italy
MELISSA ANNE TUTTY • Laboratory for Biological Characterization of Advanced Materials
(LBCAM), Trinity Translational Medicine Institute, Trinity Centre for Health Sciences,
Trinity College Dublin, Dublin, Ireland; Nanomedicine and Molecular Imaging Group,
Trinity Translational Medicine Institute (TTMI), School of Medicine, Trinity College
Dublin, Dublin, Ireland
EIRINI G. VELLIOU • Centre for 3D models of Health and Disease, Division of Surgery and
Interventional Science, University College London, London, UK
 Part I

Overview of the Existing Methods for Cancer Cell Culture

 Chapter 1

Cancer Cell Culture: The Basics and Two-Dimensional

Cultures
Melissa Anne Tutty, Sarah Holmes, and Adriele Prina-Mello

Abstract
Despite significant advances in investigative and therapeutic methodologies for cancer, 2D cell culture
remains an essential and evolving competency in this fast-paced industry. From basic monolayer cultures
and functional assays to more recent and ever-advancing cell-based cancer interventions, 2D cell culture
plays a crucial role in cancer diagnosis, prognosis, and treatment. Research and development in this field call
for a great deal of optimization, while the heterogenous nature of cancer itself demands personalized
precision for its intervention. In this way, 2D cell culture is ideal, providing a highly adaptive and responsive
platform, where skills can be honed and techniques modified. Furthermore, it is arguably the most efficient,
economical, and sustainable methodology available to researchers and clinicians alike.
In this chapter, we discuss the history of cell culture and the varying types of cell and cell lines used today,
the techniques used to characterize and authenticate them, the applications of 2D cell culture in cancer
diagnosis and prognosis, and more recent developments in the area of cell-based cancer interventions and
vaccines.

Key words Cancer cell culture, Cell line, Primary cells, Cell culture analysis, Characterization, Cancer
research

1 Introduction

1.1 Cancer Cell Lines Cell culture is a century-old and vitally important tool in cancer
research, widely used by biomedical researchers in hospitals, aca-
demic settings, and industry laboratories for many applications
[1]. The first citations of cell culture go back 125 years ago, to a
publication from Roux et al., who successfully cultured tissue from
chick embryos for several days in saline [2, 3]. Following this,
Ljunggren showed in 1898 that it was possible for the human
skin to survive in ascitic fluid ex vivo [4], and in 1907, Harrison
showed that frog embryo tissue in frog lymph clots could not only
survive but also sprout fibers [5]. Losee and Ebeling (1914) were
the first to culture cancer cells [6], with William Earle producing
the first continuous rodent cell lines at the National Cancer

Dania Movia and Adriele Prina-Mello (eds.), Cancer Cell Culture: Methods and Protocols,
Methods in Molecular Biology, vol. 2645, https://doi.org/10.1007/978-1-0716-3056-3_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

3
4 Melissa Anne Tutty et al.

Table 1
Key terms used in cancer cell culture

Term Definition
Authenticated/validated Cell line whose identity, purity, sterility, and functionality is confirmed
cell line
Cell bank Repository of cell lines
Certificate of analysis
Cell culture Growth and maintenance of tissue explants in vitro
Cell line Cells subcultured beyond their initial primary culture
Continuous cell line Cell line that has unlimited number of doublings; immortal cell culture
Clone Cells derived from one single origin cell
Confluent Cells covering all substrate
Doubling time Time taken for cells to double
Finite cell line Cell line with limited lifespan; undergoes senescence after a certain point
Lag growth phase Initial slow growth phase: Cells subculture here
Log growth phase Exponential rapid growth phase
Passage/subculture Expansion of cells from one flask to another
Plateau hase Slow growth phase; occurs when cells become confluent
Primary culture Initial culture of dissociated cells from tumors or extracted from blood
Substrate Matrix/material on which cells grow
Tissue bank/biobank Repository of human tissue samples
Tissue culture Growth and maintenance of dissociated cells in vitro
Adapted from Ref. Langdon [9]

Research Institute in 1943 [7]. Earle was closely followed by

George Gay, who in 1951 produced the first human continuous
cell line, HeLa, from the cancer patient Helen Lane (discussed in
more detail below) [3]. To date, HeLa cells are still one of the most
popular and widely used cells. In the decades following these
events, there has been a huge expansion in the use of cell lines,
and there are now thousands available, from a large number of
varying cell banks (Table 1), including commercial ones, where
cell lines can be readily purchased, research-based cell banks, and
nonprofit ones. Cell culture not only assists in the discovery of new
molecules but also elucidates their functions. It is also used for the
time-effective and high-throughput toxicity screening of new ther-
apeutic compounds. Cell culture is not only undertaken with cancer
cells but also healthy ones, and it is now commonplace to obtain
non-neoplastic cells such as endothelial or inflammatory cells for
Another Random Scribd Document
with Unrelated Content
and, at the ends of the third, and fourth, rows, leaving twenty-six
stitches unknit.
Knit a third pattern in white, leaving twenty-six stitches unknit at
the end of the first three rows; but, in the fourth row, pearl all the
stitches to the end of the needle.
Knit a fourth pattern in white;—in the first row of this, knit all the
stitches to the other end of the needle; but, at the end of the third
row, leave twenty stitches unknit. Pearl the fourth row, with the third
shade of the coloured wool, leaving twenty stitches unknit, at the
end.
Knit three more patterns in colours,—(the centre being darker
than the other two) leaving one stitch more unknit, at the end of
each row. The fourth row of the third pattern is to be pearled in
white.
Knit seven patterns in white, leaving one stitch unknit at the end
of each row; and also, omitting to make a stitch between the first
two, and last two, stitches of the second row.
When the seventh pattern is completed, there should be only
eight stitches on the needle: with these eight, knit one pattern; and,
at the end of the pearled row, pass the last stitch, before knitting, on
to the other needle; then knit it, together with two of those before
left unknit,—three in one.
Proceed in the like manner, at the end of the next, or double stitch
row, and continue the same, until seven patterns are finished, from
the above eight stitches.
Before commencing the last pearled row, fasten on the third shade
of coloured wool, and pearl to the end of the row. Then, knit the last
stitch, together with three of the unknit stitches,—four in one.
Repeat the same, at the end of the next row.
In the next pearled row, knit again four together, as above, and
three single stitches beyond, at the end of the double stitch row:
also, knit four together, and one single, and two double beyond.
 When two patterns, in the third shade, are completed, fasten on
the white wool, and pearl a row;—at the end of this, knit three
single stitches of the unknit beyond. In the next row,—knit,
alternately, a double and a single stitch, throughout the row;—
knitting the last stitch, together with one row of the unknit, and two
single ones beyond.
In the next row, make a stitch between each, as usual; and, at the
end of the next pearled row, knit three single stitches beyond. Work
the three next rows as follows:
First—three double, and one single stitch, alternately:—the last
must be a double stitch, and a single stitch beyond.
Second—a stitch between each, and three single stitches beyond.
Third—plain knitting, and three single stitches beyond.
Before commencing the next pearled row, fasten on the darkest
coloured wool; knit a pattern quite to the end of each needle, and
fasten off,—omitting the pearled row.
The cord for tieing this cap may be made by plaiting one light-
coloured, and two dark-coloured, threads of wool together; each
thread consisting of four plies of German wool. One cord passes
across the front of the cap, under the chin, and another round the
caul, with a bow at the side: the ends finishing with a tassel of white
wool.—Ribands, which are prettier, may be substituted.
 A Baby’s Hood in Plain Knitting.

Needles, No. 2.—Double German wool.

Cast on fifty stitches.—Knit eighty plain rows; sixty of which are to
be rolled up to form the front.
Sew together three inches of the cast-on part; and draw up the
remainder for the crown.
Cast on fifty stitches for the hood, and work forty plain rows.
When finished, it may be lined with white silk or satin, and
trimmed with narrow satin riband.
 A warm square Shawl.

Eight-thread fleecy, two colours, say scarlet and drab.—Needles,

No. 1.
Cast on any number of stitches that can be divided by four, with
scarlet.
First row—Knit two; knit two together;(a) bring the wool forward,
knit two; knit two together.—Repeat from (a).—Finish with—bring
the wool forward, knit four.
Second row—pearl two; pearl two together;(b) turn the wool
round the needle, pearl two; pearl two together.—Repeat from (b).—
Finish with—turn the wool round the needle, pearl four.
Work four other similar rows, making altogether six rows,—with
scarlet.
Seventh row—with drab,—knit two;(c) bring the wool forward, knit
two together; knit two.—Repeat from (c).—Finish with—bring the
wool forward, knit two together.
Eighth row—pearl two;(d) turn the wool round the needle, pearl
two together; pearl two.—Repeat from (d).—Finish with—turn the
wool round the needle, pearl two together.
Work four other rows, similar to the two last, with drab; then,
commence again, as at first row, with scarlet.
 A Shawl in Raised Knitting.

The centre may be worked in violet, or dark claret: the border in

eight shades of stone colour, including the extreme shades—black
and white.—German wool.—Two needles, No. 16, and one needle,
No. 10.
Commence with the border, by casting on four hundred and eighty
stitches, with black. Work two patterns in each shade of colour,
decreasing, by knitting two together, at the beginning of each row,
except on the first four;—when four hundred and twenty stitches will
remain on the needle for the centre of the shawl.
With the violet or claret wool, work a square of four hundred and
twenty stitches, to form the centre. Then,—
Commence the opposite border, with the white shade of stone
colour, increasing on every row, except on the last four; and
reversing the shades of colour, so as to form a similar border to the
first.
Two side borders, in separate pieces, are now to be worked in the
manner first described. These are afterwards to be sewn on;—the
decreasing having formed an angle, that admits of the right shades
of colour joining correctly.
Each pattern of the raised knitting is composed of four rows,
which are worked as follows;—
First row—with small needle,—bring the wool forward, knit two
together.—Repeat.
Second row—with large needle,—plain knitting.
Third row—with small needle,—plain knitting.
Fourth row—with small needle,—pearl knitting.
 A Vandyke Edging.

Cast on twelve stitches.—Needles, No. 26.—Cotton, No. 20.

First row—slip one; knit two; bring the cotton forward, knit two
together; bring the cotton forward twice, knit two together; knit five.
Second row—slip one; knit six; pearl one; knit two; bring the
cotton forward, knit two together; knit one.
Third row—slip one; knit two; bring the cotton forward, knit two
together; knit two; bring the cotton forward twice, knit two
together; knit four.
Fourth row—slip one; knit five; pearl one; knit four; bring the
cotton forward, knit two together; knit one.
Fifth row—slip one; knit two; bring the cotton forward, knit two
together; knit four; bring the cotton forward twice, knit two
together; knit three.
Sixth row—slip one; knit four; pearl one; knit six; bring the cotton
forward, knit two together; knit one.
Seventh row—slip one; knit two; bring the cotton forward; knit
two together; knit ten.
Eighth row—slip one; knit one, pass the slip-stitch over it; knit
one, pass the stitch over it; knit one, pass the stitch over it; knit
eight; bring the cotton forward, knit two together; knit one.
Commence again, as at first row.
 Insertion Leaf-Pattern for Tidies

Cast on twenty-one stitches for each pattern.—Needles, No. 18—

Cotton, No. 14.
First row—knit one; knit two together; bring the thread forward
twice, knit two together; bring the thread forward, knit two together,
taken at the back; knit three; pearl one; knit three; knit two
together; bring the thread forward, knit two together; bring the
thread forward twice, knit two together; knit one.
Second row—knit three; pearl one; knit one; pearl one; turn the
thread round the needle, pearl two together; pearl two; knit one;
pearl two; pearl two together; turn the thread round the needle,
pearl one; knit one; pearl one; knit three.
Third row—knit one; knit two together; bring the thread forward
twice, knit two together; knit two; bring the thread forward, knit two
together, taken at the back; knit one; pearl one; knit one; knit two
together; bring the thread forward, knit two; knit two together;
bring the thread forward twice, knit two together; knit one.
Fourth row—knit three; pearl one; knit one; pearl three; turn the
thread round the needle, pearl two together; knit one; pearl two
together; turn the thread round the needle, pearl three; knit one;
pearl one; knit three.
Fifth row—knit one; knit two together; bring the thread forward
twice, knit two together; knit four; bring the thread forward, knit
three together; bring the thread forward, knit four; knit two
together; bring the thread forward twice, knit two together; knit
one.
Sixth row—knit three; pearl one; knit one; pearl five; knit one;
pearl five; knit one; pearl one: knit three.
Commence again, as at first row.
 A very pretty insertion may be made, by knitting one pattern only
of the above.
 Vandyke and Open Pattern for a Tidy, etc.

Cast on twelve stitches for each pattern, and six, for the three
edge stitches on either side.—Needles, No. 18.—Cotton, No. 14.
N.B. To prevent confusion, the edge stitches are omitted in the
directions.
First row—knit one; bring the thread forward, knit two together,—
four times; knit three.—Repeat.
Second row—pearl knitting.
Third row—knit two; bring the thread forward, knit two together,—
four times; knit two.—Repeat.
Fourth row—pearl knitting.
Fifth row—knit three; bring the thread forward, knit two together,
—four times; knit one.—Repeat.
Sixth row—pearl knitting.
Seventh row—knit four; bring the thread forward, knit two
together,—four times.—Repeat.
Eighth row—pearl knitting.
Ninth row—knit three; bring the thread forward, knit two together,
—four times; knit one.—Repeat.
Tenth row—pearl knitting.
Eleventh row—knit two; bring the thread forward, knit two
together,—four times; knit two.—Repeat.
Twelfth row—pearl knitting.
Commence again, as at first row.
 Open Stripe Pattern for a Tidy, etc.

Cast on twenty-four stitches for each pattern.—Needles, No. 18.—

Cotton, No. 14.
First row—knit two together,—four times; bring the thread
forward, knit one,—seven times; bring the thread forward, knit two
together; knit two together,—three times; knit one.—Repeat.
Second row—pearl knitting.
Repeat these two rows, until a piece be worked of the required
size; then,—finish by casting off every twenty-three stitches, and
dropping every twenty-fourth stitch, to form the open stripe.
 Another Tidy.

Cast on six stitches for each pattern, and four, for two edge
stitches on either side.—Needles, No. 18.—Cotton, No. 14.
N.B. To prevent confusion, the edge stitches are omitted in the
directions.
First row—knit two together; knit two; bring the thread forward,
knit two together; bring the thread forward.—Repeat.
Second row—pearl knitting.
Repeat these two rows, three times.
Ninth row—knit three;(a) bring the thread forward, knit two
together; bring the thread forward, knit two; knit two together.—
Repeat from (a).
Tenth row—pearl knitting.
Repeat the last two rows, twice; and commence again, as at first
row.
 Feather Pattern for a Tidy.

Cast on nineteen stitches for each pattern, and four over, for two
stitches at each edge.—Needles, No. 22.—Cotton, No. 18.
First row—plain knitting.
Second row—bring the thread forward, knit one,—three times;
knit two together,—three times; knit one; knit two together,—three
times; bring the thread forward, knit one,—three times.—Repeat.
Third row—pearl knitting.
Fourth row—plain knitting.
Fifth row—pearl knitting.
Commence again, as at second row.
 Small Rose-leaf Pattern for a Tidy.

Cast on any number of stitches that can be divided by six, adding

four stitches over, to keep the pattern even at the beginning and
end.—Needles, No. 18.—Cotton, No. 14.
First row—knit one; knit two together, taken at the back;(a) bring
the thread forward, knit one; bring the thread forward, knit one; slip
one; knit two together, pass the slip-stitch over them; knit one.—
Repeat from (a).—Finish with—bring the thread forward, knit two
together.
Second row—pearl knitting.
Third row—knit two;(b) bring the thread forward, knit three; bring
the thread forward, slip one; knit two together, pass the slip-stitch
over them.—Repeat from (b).—Finish with—bring the thread
forward, knit three.
Fourth row—pearl knitting.
Fifth row—knit two;(c) bring the thread forward, knit one; slip
one; knit two together, pass the slip-stitch over them; knit one; bring
the thread forward, knit one.—Repeat from (c).—Finish with—bring
the thread forward, knit two together, taken at the back.
Sixth row—pearl knitting.
Seventh row—knit two together; knit one;(d) bring the thread
forward, slip one; knit two together, pass the slip-stitch over them;
bring the thread forward, knit three.—Repeat from (d).—Finish with
—bring the thread forward, knit three.
Eighth row—pearl knitting.
Commence again, as at first row.
 Point de l’Echelle, for a Tidy, etc.

Cast on thirty-six stitches for every two patterns, and one over, for
the centre or dividing stitch between each pattern.—Needles, No. 16.
—Cotton, No. 12.
First row—pearl three;(a) pass the thread over, knit three; knit two
together; knit three; knit two together; knit three; bring the thread
forward, pearl five.—Repeat from (a).—Finish with—pearl three.
Second row—pearl knitting, except the stitches pearled in the
previous row, which are to be knitted plain.
Third row—pearl three;(b) knit one; bring the thread forward, knit
three; knit two together; knit one; knit two together; knit three;
bring the thread forward, knit one; pearl five.—Repeat from (b).—
Finish with—pearl three.
Fourth row—same as second.
Fifth row—pearl three;(c) knit two; bring the thread forward, knit
three; knit three together; knit three; bring the thread forward, knit
two; pearl five.—Repeat from (c).—Finish with—pearl three.
Sixth row—the same as second.
Commence again, as at first row.
N.B. In the above pattern, when casting off, every nineteenth
stitch is to be dropped.
 A Fish or Basket Napkin.

Cast on twenty-four stitches for each pattern, and one over, for
the edge stitch.—Needles, No. 16.—Cotton, No. 12.
First row—knit one; bring the thread forward, knit four; knit two
together, taken at the back; pearl eleven; knit two together; knit
four; bring the thread forward.—Repeat.
Second row—pearl two; turn the thread round the needle, pearl
four; pearl two together; knit nine; pearl two together, taken at the
back; pearl four; turn the thread round the needle, pearl one.—
Repeat.
Third row—knit three; bring the thread forward, knit four; knit two
together, taken at the back; pearl seven; knit two together; knit
four; bring the thread forward, knit two.—Repeat.
Fourth row—pearl four; turn the thread round the needle, pearl
four; pearl two together; knit five; pearl two together, taken at the
back; pearl four; turn the thread round the needle, pearl three.—
Repeat.
Fifth row—knit one; bring the thread forward, knit eight; knit two
together, taken at the back; pearl three; knit two together; knit
eight; bring the thread forward.—Repeat.
Sixth row—pearl two; turn the thread round the needle, pearl
eight; pearl two together; knit one; pearl two together, taken at the
back; pearl eight; turn the thread round the needle, pearl one.—
Repeat.
Seventh row—knit three; bring the thread forward, knit eight; knit
two together, twice; knit seven; bring the thread forward, knit two.—
Repeat.
Eighth row—pearl four; turn the thread round the needle, pearl
seven; slip one; pearl two together, pass the slip-stitch over them;
pearl seven; turn the thread round the needle, pearl three.—Repeat.
Commence again, as at first row.
 Fern Pattern Fish Napkin.

Cast on twenty-nine stitches for each pattern, and nine over, to

make both sides correspond.—Needles, No. 16.—Cotton, No. 12.
N.B. The nine extra stitches, at the end of each row, are to be
knitted the same as the nine stitches, at the commencement.
First row—pearl three; knit one; bring the thread forward twice,
knit two together; pearl three; pass the thread over, knit five; knit
two together; knit six; knit two together; knit five; bring the thread
forward.—Repeat.
Second row—knit three; pearl two together; slip one; turn the
thread round the needle, pearl one; knit three; pearl one; turn the
thread round the needle, pearl five; pearl two together; pearl four;
pearl two together; pearl five; turn the thread round the needle,
pearl one.—Repeat.
Third row—pearl three; knit one; bring the thread forward, slip the
loops; knit one; pearl three; knit two; bring the thread forward, knit
five; knit two together; knit two; knit two together; knit five; bring
the thread forward, knit two.—Repeat.
Fourth row—knit three; pearl one; knit the loops; pearl one; knit
three; pearl three; turn the thread round the needle, pearl five; knit
two together,—twice; pearl five; turn the thread round the needle,
pearl three.—Repeat.
Commence again, as at first row.
 Open Pattern for a small Quilt.

Cast on five stitches for each pattern.—German wool, used double.

—Needles, No. 7.
First row—bring the thread forward, slip one; knit two, pass the
slip-stitch over them; bring the thread forward, slip one; knit one,
pass the slip-stitch over it.—Repeat.
Second row—pearl knitting.
Commence again, as at first row.
This quilt is prettiest when lined with coloured silk. It should be
trimmed with a vandyke edging, knitted with wool.
 Pretty Open Stitch for a Bag.

Middle-sized netting silk.—Needles, No. 23.

Cast on six stitches for each pattern.—Forty-four patterns form an
ordinary sized bag.
First round—bring the silk forward, slip one; knit two together,
pass the slip-stitch over them; bring the silk forward, knit three.—
Repeat.
Second round—plain knitting.
Third round—knit three;(a) bring the silk forward, slip one; knit
two together, pass the slip-stitch over them; bring the silk forward,
knit three.—Repeat from (a).—Finish with—bring the silk forward,
slip one; knit two, pass the slip-stitch over them.—The latter
operation prevents the round decreasing.
Fourth round—plain knitting.
Commence again, as at first round.
 A new Pence Jug or Purse.

Two colours in German wool,—say blue and orange.—Needles, No.

25.
Cast twelve stitches on the first needle, for the spout; and ten
stitches on each of three other needles,—with blue. Knit three
rounds.
Fourth round—with orange, plain knitting.
In the next twelve rounds, pearl two, and knit two, alternately;
except the twelve stitches for the spout, which are to be knitted
plain; decreasing one stitch, alternately, on each side of these
twelve, in the first four rounds, and one stitch on one side only, in
the next eight rounds. The spout, together with the first seventeen
rounds, will now be finished, when thirty-one stitches should remain
on the needles.
Knit twelve rounds, pearling two, and knitting two, alternately,
except under the spout, where one stitch only is to be knitted. Then,
—
With the blue wool, commence forming the bowl of the jug;
increasing, by knitting two stitches in one,—first knitting the front of
the stitch, and then the back; which will prevent the appearance of
the increasing.
Pearl two rounds.
Knit four rounds,—with orange, slipping every fourth (blue) stitch.
Knit one round,—with blue, increasing twelve stitches, by knitting
two stitches in one, as before, every sixth stitch.
Pearl two rounds,—with blue.
Knit four rounds,—with orange, slipping every fourth (blue) stitch,
as before.
 Knit one round,—with blue, increasing twelve stitches, by knitting
two stitches in one, as before; when eighty-eight stitches should
count on the needles.
Pearl two rounds,—with blue.
Knit five rounds,—with orange.
Knit one round,—with blue.
Pearl two rounds,—with blue.
Knit four rounds,—with orange, slipping every fourth (blue) stitch,
as before.
Knit one round, decreasing ten stitches,—with blue.
Pearl two rounds,— with blue.
Knit four rounds,—with orange, slipping every fourth (blue) stitch,
as before.
Knit one round, decreasing six stitches,—with blue.
Pearl two rounds,—with blue.
Seventy-two stitches should now remain on the needles. Divide
this number of stitches by six, and knit eleven rounds with orange;
decreasing six stitches in each round, by knitting two together, at
the commencement of each division; when a star of six points will be
formed, and six stitches only will remain on the needles:—these are
to be drawn up at the point.
Take up eight stitches on the side opposite the spout; then, in
pearled, and plain rows, work a piece about an inch and a half long
—with orange. The end of this is to be attached to the first row of
the bowl of the jug, to form the handle.
 An Easy Stitch for various Purposes.

This is an easy and light stitch. It may be worked with fine or

coarse wool, for half handkerchiefs, bonnet caps, under spencers,
sleeves, etc. For most useful purposes, three-thread fleecy, and
needles No. 12, may be employed.
Cast on any number of stitches that can be divided by three.
First row—bring the wool forward, knit two together; knit one.—
Repeat.
Second row—plain knitting.
Repeat these two rows, alternately.
When knitting sleeves, or any other article, where decreasing is
necessary, knit two together, at the beginning of every second row.
 A Chancelière.

Six-thread fleecy, say two colours, blue and drab.—Needles, No.

14.
Cast on forty-two stitches, and knit six, or eight, patterns, (see
directions next page), alternately drab and blue, until a piece about
twenty-four inches in length be worked. Then—with one colour—
knit, in a similar manner, a band about thirty inches in length, and
three or four inches wide, to form the sides.
In brioche stitch, work two corresponding pieces, with white, to
form the lining. These four pieces are to be wadded, and mounted,
in the usual form. The opening for the feet should be edged with a
worsted ermine trimming.
The pattern is knitted as follows;—
Cast on any number of stitches that can be divided by three.
First row—bring the wool forward, slip one; knit two together.—
Repeat.
Second row—knit two; slip the long stitch.—Repeat.
Third row—knit two together;(a) bring the wool forward, slip one;
knit two together.—Repeat from (a).—Finish with—bring the wool
forward, knit one.
Fourth row—knit one; slip one;(b) knit two; slip one.—Repeat from
(b).
Commence again, as at first row.
 A Warm Half-square Shawl.

German wool—used double.—Needles, No. 6.—Scarlet, and four

shades of bright green slate colour, form a pretty contrast. The
shawl should be worked in alternate stripes of the two colours; each
stripe being composed of four patterns,—that is, one pattern in each
shade of the green slate colour, and four patterns in scarlet.
Cast on two hundred and forty stitches with the lightest shade of
slate colour.
First row—bring the wool forward, slip one; knit two, pass the slip-
stitch over them.—Repeat to the end of the row, except on the two
last stitches, which are to be knitted together.
Second row—pearl knitting.
Repeat these two rows, with the three other shades of slate
colour,—decreasing (by knitting two together) at the end of every
pattern row.
With the scarlet wool, knit four patterns, in a similar manner.
Two stripes, of an equal width, will thus be formed;—the one,
composed of four shades of slate colour, and the other, of scarlet.
These are to be repeated, alternately, until the half square be
finished.
A fringe for this shawl may be knitted with scarlet, as follows.
 Fringe for a Shawl.

This forms a very pretty fringe for the preceding shawl pattern.
Cast on twelve stitches with scarlet German wool—used double.—
Needles, No. 6.
First row—bring the wool forward, knit two together.—Repeat.
Every row is the same. When finished, cast off three patterns; and
unravel the remainder for the fringe.
Before unravelling, the knitting should be thoroughly damped, and
afterwards dried before a fire. This will cause the wool to curl, and
form a better fringe. Each loop should be knotted, close to the
knitting.
It is advisable to knit this fringe in one length. It may be easily
made to sit well round the point of the shawl, by fulling it a little, in
the sewing on.
THE TWELVE FOLLOWING PATTERNS ARE INTENDED FOR A SET OF D’OYLEYS.—
THEY ARE TO BE WORKED WITH KNITTING COTTON, NO. 20, AND NEEDLES,
NO. 26. WHEN WORKED WITH COARSER COTTON, AND LARGER NEEDLES, THE
OPEN PATTERNS FORM TIDIES; AND THE CLOSE PATTERNS (OF THE SIZE
DIRECTED), MAKE PRETTY COVERS FOR TOILET CUSHIONS, ETC.
 D’Oyleys.

These D’Oyleys, as nearly as the patterns will allow, work about

six inches square; they have an open border, with a plain edge of
three stitches, on each side. Some of the centres are arranged to
contain a certain number of perfect patterns; but others, where such
an arrangement would have interfered with the symmetry of the
borders, are curtailed of a few stitches:—thus, No. IV. contains four
perfect patterns, each composed of fifteen stitches; and one pattern
of thirteen stitches only—the open part of the pattern, on one side,
being omitted.
In addition to the borders above mentioned, these D’Oyleys
should be trimmed with a netted scalloped edging, or, with one of
the knitted edgings to be found in this book: a coarse lace, however,
if preferred, will equally answer the purpose.
As the first twelve rows—composing the border—of each D’Oyley,
are the same, the repetition of them, in each pattern, appears
useless; the following directions, therefore, will serve for the
commencement of all, and form—
 A Border for each D’Oyley.

Knit six plain rows.

Seventh row—knit three; bring the thread forward, knit two
together.—Repeat, except on the last three stitches, which knit plain.
Eighth row—pearl knitting, except on the first three, and last
three, stitches, which are to be knitted plain.
Repeat the seventh, and eighth, rows, making altogether, twelve
rows from the commencement.
The above forms the upper border of the D’Oyley. The side
borders are included in the directions given for each row of the
centre patterns.—The lower border—worked the same (reversed) as
the upper—it appears unnecessary to repeat.
N.B. The pattern of the centre is always commenced on the
thirteenth row of each D’Oyley.
 I.
Willow Pattern.

Cast on seventy-four stitches; that is,—eighteen for the two

borders, and fifty-six for the centre. Each centre pattern occupies
four stitches.
Knit the upper border, as before directed. Commence the side-
borders, and centre, as follows;—
First row—knit three; bring the thread forward, knit two together,
—three times; knit two; knit two together;(a) bring the thread
forward, knit two; knit two together.—Repeat from (a).—Finish with
—bring the thread forward, knit four; bring the thread forward, knit
two together,—three times; knit three.
Second row—knit three; pearl eight; pearl two together;(b) turn
the thread round the needle, pearl two; pearl two together.—Repeat
from (b).—Finish with—turn the thread round the needle, pearl ten;
knit three.
Repeat these two rows twice,—making in all six rows.
Seventh row—knit three; bring the thread forward, knit two
together,—three times; knit two;(c) bring the thread forward, knit
two together; knit two. Repeat from (c).—Finish with—bring the
thread forward, knit two together,—four times; knit three.
Eighth row—knit three; pearl eight;(d) turn the thread round the
needle, pearl two together; pearl two.—Repeat from (d).—Finish with
—turn the thread round the needle, pearl two together; pearl six;
knit three.
Repeat the seventh and eighth rows,—twice, making from the
commencement of the pattern—twelve rows. Then,—
Commence again, as at first row,—working a sufficient number of
rows to form a square; then repeat the border.